The effect of starvation on the control of phosphofructokinase activity in the epithelial cells of the rat small intestine.

1. The effect of depriving rats of food for 48 h on the specific activity of phosphofructokinase in the epithelial cells of the small intestine and on the regulatory properties of the enzyme displayed in crude (particle-free) mucosal extracts was studied. 2. The specific activity of phosphofructokinase, measured under optimal conditions at pH8, in the mucosa of fed rats showed a negative aboral gradient along the intestine, decreasing from 15.2 +/- 1.2 units (mumol/min)/g wet wt. in the proximal jejunum to 4.6 +/- 1.2 units/g wet wt. in the terminal ileum. 3. After starvation, the gradient was diminished, but not abolished; the diminution in gradient was due almost exclusively to a decrease in the specific activity of phosphofructokinase in the proximal jejunum by about 30%, there being no change in the terminal ileum. 4. In fed rats, the susceptibility of phosphofructokinase to inhibition by ATP, when assayed in crude mucosal extracts under suboptimal conditions, was independent of length along the small intestine; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM-fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8, v0.5/V, was 0.36 +/- 0.05 in the proximal jejunum and 0.42 +/- 0.07 in the terminal ileum. 5. After starvation, the susceptibility of phosphofructokinase to inhibition by ATP was increased and was again found to be independent of length along the small intestine: after starvation, v0.5/V was 0.19 +/- 0.04 and 0.20 +/- 0.07 for the proximal jejunum and the terminal ileum respectively. 6. Re-feeding of previously starved rats on a high-carbohydrate diet overnight for 16 h restored both the specific activities of phosphofructokinase and its susceptibility to inhibition by ATP to normal values for fed rats. 7. The data support the idea that the specific activities and the regulatory properties of phosphofructokinase in the epithelial cells of rat small intestine are mediated by distinct humoral factors. 8. The changes in glucose utilization rate of the jejunum when rats are starved can in principle be accounted for by a combination of changes in the specific activity and in the regulatory properties of mucosal phosphofructokinase.     

Stimulation of glycolysis and accumulation of a stimulator of phosphofructokinase in hepatocytes incubated with vasopressin.

Vasopressin stimulates glycolysis in hepatocytes prepared from fed rats, or from starved rats when incubated with glucose. It causes the stimulation of phosphofructokinase activity and the accumulation of a stimulator of phosphofructokinase, which is probably fructose 2,6-bisphosphate, the recently discovered stimulatory of phosphofructokinase.     

The regulation of lipogenesis in vivo in the lactating mammary gland of the rat during the starved-refed transition. Studies wtih acarbose, a glucosidase inhibitor.

Depression of carbohydrate digestion by oral administration of acarbose, a glucosidase inhibitor, led to a 75% inhibition of the re-activation of lipogenesis in vivo in the mammary gland of 18 h-starved lactating rats refed with 5 g of chow diet. Rates of [1-14C]glucose incorporation in vitro into lipid and CO2 in mammary-gland acini isolated from refed animals were elevated compared with acini from starved rats, but acarbose treatment completely prevented this stimulation. Gastric intubation of glucose led to a large stimulation of lipogenesis in the mammary gland of starved lactating rats, similar to that induced by refeeding with chow diet; this was dependent on the amount of glucose given and the time elapsed between glucose administration and injection of 3H2O for the measurement of lipogenesis. The switch-on of lipogenesis in the mammary gland of starved lactating rats, by refeeding or by intubation of glucose, was associated with a decrease in the ratio of [glucose 6-phosphate]/[fructose 1,6-bisphosphate] in the gland, indicative of an increase in phosphofructokinase activity. A time-course study revealed that the ratio decreased rapidly over the first 30 min of chow refeeding, after which a large surge in lipogenesis was seen. Acarbose, given 25 min after the onset of refeeding, led to a stepwise increase in the ratio, in parallel with the observed decrease in lipogenic activity. It is concluded that the control of lipogenesis in the mammary gland is closely linked to the availability of dietary carbohydrate. An important site of regulation of lipogenesis in the gland appears to be at the level of phosphofructokinase. A possible role of insulin in the regulation of phosphofructokinase activity, and the acute modulation of insulin-sensitivity in the gland during the starved-refed transition, are discussed.     

Effects of starvation and streptozotocin-induced diabetes on the activity of phosphofructokinase in the epithelial cells of rat colon

The regulation of phosphofructokinase in the colonic mucosa of 48 h-starved and streptozotocin-induced diabetic rats was investigated. The specific activities of phosphofructokinase from colonic mucosa of starved and diabetic rats were found to be diminished compared with normal controls. The enzyme obtained from the colonic mucosa of normal, diabetic and starved rats showed sigmoidal velocity curves with respect to fructose-6-phosphate, with apparent Km values of 0.6, 0.62 and 0.7 mM, respectively. However, the present results indicated that phosphofructokinase from the epithelial cells of rat colon is not regulated in a manner similar to that of the intestinal enzyme, which was shown to be highly regulated.     

The nature of the decreased activity of glycogen synthase phosphatase in the liver of the adrenalectomized starved rat

We have investigated the nature of the decrease in synthase phosphatase activity which occurs progressively in the livers of adrenalectomized rats that are starved for 48h. No evidence could be found for the accumulation of an inhibitor. Addition of the heat-stable deinhibitor protein, which antagonizes the effects of thermostable inhibitor proteins (inhibitor-1 and modulator), did not affect the activity of synthase phosphatase in gel-filtered liver extracts from normal or adrenalectomized starved rats; it did, however, increase the activity of phosphorylase phosphatase about fivefold in either condition. The restoration of synthase phosphatase activity by cortisol in vivo was prevented by actinomycin D. Further evidence concerning the nature of the missing protein came from a comparison of synthase phosphatase activities in liver homogenates from control and adrenalectomized starved rats, with the use of three distinct synthase b substrates. The apparent loss of synthase phosphatase activity in the deficient homogenates varied between 30% and 90% according to the type of substrate. The magnitude of this decrease corresponds to the degree of dependence of these substrates on the G-component of synthase phosphatase for efficient conversion to the alpha-form. No G-component could be isolated from livers of adrenalectomized starved rats. Cross-combination of subcellular fractions from control and deficient livers revealed an almost total loss of G-component, with little loss of S-component. This specific loss of functional G-component is identical to the deficiency previously observed in the livers of rats with severe chronic alloxan-diabetes.     

Decreased phosphofructokinase activity in skeletal muscle of diabetic rats

The activities of phosphofructokinase, aldolase and pyruvate kinase were diminished in extracts from skeletal muscle of streptozotocin diabetic rats, whereas the activities of glucose phosphate isomerase and phosphoglucomutase were not changed. Treatment of diabetic rats with insulin restored the activity of phosphofructokinase to normal. A kinetic study of the partially purified enzyme from normal and diabetic rats showed identical Michaelis constants for ATP and equal sensitivity to inhibition by excess of this substrate. Extracts of quick frozen muscle from diabetic rats had higher levels of citrate (an inhibitor of phosphofructokinase) and lower levels of D-fructose-1,6-bisphosphate and D-glucose-1,6-bisphosphate (activators of this enzyme). The levels of D-fructose-6-phosphate, D-glucose-6-phosphate, ATP, ADP and AMP were the same for the two groups. Our data suggest that the in vivo decrease of phosphofructokinase activity in skeletal muscle of diabetic rats is due to a decrease in the level of the enzymatically active protein as well as to an unfavorable change in the level of several of its allosteric modulators.     

Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle.

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Fructose 2,6-bisphosphate in livers of genetically obese rats.

Livers of genetically obese (fa/fa) rats, starved for 24 h, contained more fructose 2,6-bisphosphate, glucose 6-phosphate, fructose 6-phosphate and glycogen, and more pyruvate kinase and phosphofructokinase 2 activities, than livers of control lean rats. These changes may be explained in terms of cyclic AMP concentration, which was decreased in livers of obese starved rats.     

Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition

Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.     

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.     

The effects of haem on translational control of protein synthesis in cell-free extracts from fed and lysine-derived Ehrlich ascites tumour cells

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit X Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.     

The effect of thermal injury on the regulation of phosphofructokinase in the mucosa of rat small intestine

The effects of thermal injury (72 h post-injury), 72 h-partial (20% less food) or full starvation on the regulation of phosphofructokinase in the mucosa of rat small intestine were studied. Thermal injury and 72 h-partial or full starvation decreased the activity ratio v0.5/V, but the ratios obtained for thermally injured or fully starved rats were significantly lower than those of controls or partially starved rats. The susceptibility of phosphofructokinase to ATP inhibition was increased after thermal injury and 72 h-partial or full starvation compared to that of normal controls. However, these changes that occurred in the enzyme activities of the rat small intestine were mainly specific to injury per se but do not exclude the contribution of partial starvation during the same period of time.     

Antizyme to ornithine decarboxylase is present in the liver of starved rats.

Antizyme to ornithine decarboxylase (ODC) and ODC-antizyme complex were both present in liver cytosols of starved rats. The antizyme was identified by its molecular weight, kinetic properties, formation of a complex with ODC, and reversal of its inhibition by antizyme inhibitor. The average amount of antizyme in liver cytosols of starved rats was 0.1 unit/mg of protein, roughly corresponding to basal hepatic ODC activity in rats fed ad libitum. The presence of ODC-antizyme complex was detected by using antizyme inhibitor. These results indicate that antizyme participates in the regulation of ODC activity in vivo under physiological conditions.     

Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria.

Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.     

Rat-liver-type phosphofructokinase mRNA. Structure, tissue distribution and regulation.

We have cloned a full-length cDNA for rat-liver-type phosphofructokinase. The similarities of the rat liver-type phosphofructokinase mRNA to the human and mouse counterparts were 94% and 99% in their amino acid sequences and 88% and 94% in the nucleotide sequences of their coding regions, respectively. Rat liver-type phosphofructokinase mRNA was expressed in all tissues examined, but its level was regulated tissue-specifically. The nutritional and hormonal regulations of the mRNA in the liver were examined in comparison with those of two other key glycolytic enzymes, glucokinase and L-type pyruvate kinase. The level of liver-type phosphofructokinase mRNA was essentially unchanged by starvation (72 h) or diabetes. The mRNA level also did not change significantly on refeeding starved rats on a high carbohydrate diet, or treating diabetic ones with insulin. These results suggested that rat liver-type phosphofructokinase mRNA in the liver was not under control of diet or insulin, in contrast to glucokinase and L-type pyruvate kinase.     

Control in vivo of rat liver phosphofructokinase by glucagon and nutritional changes.

Glucagon (250 microgram/kg body wt.) intravenously injected into normal fed rats produces within 5 min a marked inactivation of liver phosphofructokinase, only observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate. Since half-maximal inactivation is observed at a dose of glucagon of 0.32 microgram/body wt., a dose within the range of the physiological concentrations of the hormone, the inactivation of phosphofructokinase can occur in vivo in response to physiological changes in the concentration of glucagon. In gluconeogenic conditions (starved rats or high-protein-diet-fed rats), there is a marked inactivation of liver phosphofructokinase at subsaturating concentrations of fructose 6-phosphate similar to that found in normal fed rats after glucagon treatment. In these gluconeogenic conditions a 50% decrease in the Vmax. of the enzyme is also observed. No significant changes in phosphofructokinase activity either at subsaturating concentrations of fructose 6-phosphate or in the Vmax. of the enzyme are observed when rats are fed on a high-carbohydrate diet. In the last dietary condition, glucagon treatment produces similar effects to that described in the normal fed rats. Similar results have been obtained in the above condtions for pyruvate kinase L activity when measured at subsaturating concentrations of phosphoenolpyruvate.     

Study of the fructose 6-phosphate/fructose 1,6-bi-phosphate cycle in the liver in vivo.

1. The method proposed by Rognstad & Katz [(1976) Arch, Biochem, Biophys, 177, 337-345] for the determination of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle by the randomization of carbon between C-1 and C-6 of glucose glucose formed from [1-14C] galactose was applied to anaesthetized rats and conscious mice. 2. It was checked that the hydrolysis of fructose 6-phosphate by glucose 6-phosphatase is too weak to invalidate the method. The participation of the Cori cycle in the randomization was negligible within the short experimental period used (2-4 min). 3. No detectable randomization of carbon was observed in starved animals, indicating that phosphofructokinase is inactive in this experimental condition. 4. Randomization of carbon was detected as soon as 1 min after administration of [1-14C] galactose to fed animals and was maximal at about 3-4 min. It was calculated that on average 15% of the glucose formed by the liver to fed rats was recycled through the triose phosphates. The extent of cycling was quite variable. Recycling was also observed in starved rats in which glucose had been administered intravenously 10 min previously. In these animals, recycling was completely inhibited by glucagon. 5. The main factors that appear to be responsible for the very large changes in recycling observed in various experimental conditions are the concentrations of fructose 1,6-bisphosphate and of fructose 6-phosphate and also the affinity of phosphofructokinase for fructose 6-phosphate. The concentration of nucleotides does not seem to play a role.     

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.     

Increased protein synthetic capacity in vitro of rat liver rough endoplasmic reticulum following starvation

There is a dramatic rise in the protein synthetic activity of rough endoplasmic reticulum (RER) of liver from starved and starved-refed rats, compared to normal controls. This observation is similar to the doubling of protein synthetic activity of rat liver RER observed 20 hours after partial hepatectomy (1). Oxidised glutathione (GSSG), a potent inhibitor of protein synthesis by normal RER, was a much less effective inhibitor when assayed with RER from the livers of starved rats, this again is in parallel with our earlier observations in regenerating liver. This common pattern of response is discussed in terms of a cellular control system which responds to changes in the cytoplasmic sulphydryl concentration.