Suppression of beta-oxidation restores pyruvate inhibition of pyruvate dehydrogenase kinase in starved rat heart.

Kinetic behaviour of rat heart pyruvate dehydrogenase kinase (PDHK alpha) was studied in the multi-enzyme complex (PDC) contained in two preparations: mitochondria (mPDC) and a high speed pellet of Triton-extracted tissue (hPDC). Two parameters were evaluated: Vav, related to Vmax, and Fractional Pyruvate Inhibition (FPI). Starvation of rats for 48 h led to a rise in Vav and a fall in FPI. Injection into starved rats of agents which reduce beta-oxidation of fatty acids restored, in succession, FPI and then Vav, of hPDC, to levels found in hPDC from fed animals. In vitro incubation at 30 degrees C of hPDC from starved animals restored FPI, but not Vav to 'fed' values; both were restored during in vitro incubation of mPDC from starved animals within the same time frame as in the in vivo experiments. A sharp increase of FPI, but not Vav, of hPDC from both fed and starved rats was observed in later experiments. This could have been due to differential selection of the two genes for isoenzymes of PDHK alpha proposed by other workers.

Starvation reduces pyruvate dehydrogenase phosphate phosphatase activity in rat kidney.

Pyruvate dehydrogenase complex (PDC) from rat kidney or pig heart previously inactivated by phosphorylation (PDHP) was activated in vitro by PDHP phosphatase from kidneys of starved or fed rats. Starvation for 48 h of the rats from which the PDC was prepared led to a decrease in the rate of activation of PDC at early time periods (< 2 min), particularly at submaximal concentrations of Mg2+. Using intact permeable kidney mitochondria incubated for 15 sec, it was found that starvation of rats more than doubled the Mg2+ concentration at which the half maximal increment of PDC activity (PDCa) was observed. Reduction of PDHP phosphatase activity due to starvation was also apparent when phosphatase was separated from PDC and recombined with PDC from the same or different animals. Intraperitoneal injection of insulin and glucose 1 h before sacrifice of starved rats prevented the reduction of PDHP phosphatase activity whether or not protein synthesis was inhibited. The effect of insulin in restoration of PDHP phosphatase activity of starved rats was not mimicked by 5-methylpyrazole 3-carboxylic acid, an inhibitor of lipolysis. When renal PDHP phosphatase was incubated with pig heart PDC in the presence of 10 mM Mg2+ and 0.1 mM Ca2+ the increment in PDCa, in 1 min was 30% of fully activated PDC activity (PDCt) observed after 15 min. Removal of divalent cations did not affect the increment in 1 min but prevented further increments. Conversely okadaic acid diminished 1 min increment but did not disturb PDCt. It is suggested that the different behaviour of renal PDC from fed and starved animals may partly be due to different divalent cation independent PDHP phosphatase activity.

Effects of pyruvate on pyruvate dehydrogenase kinase of rat heart.

Sensitivity of rat heart pyruvate dehydrogenase kinase (PDHK) to pyruvate inhibition was tested under various conditions using pyruvate dehydrogenase complex (PDC) in mitochondria (mPDC) and in a high speed precipitate of whole tissue homogenates (hPDC). In the latter preparation pyruvate in the range of concentration 1-10 mM caused increasing inhibition of PDHK when the enzyme was prepared from animals fed ad libitum but had no effect when the enzyme was prepared from 48 h starved animals. Similar behaviour was observed in mPDC from fed and starved animals when rotenone was present, pyruvate at 1 mM concentration stimulated PDHK from hearts of fed animals but was without effect at 10 mM. When mPDC or hPDC from hearts of starved animals was incubated at 30 degrees C for 30 min, inhibition of PDHK by pyruvate was restored.

Changes in red cell insulin receptors during recovery from severe malnutrition.

Red cell insulin binding was studied in 13 Jamaican children (age range 4-24 months), while malnourished (MAL), during early recovery (GI), late recovery (GII), and after anthropometric recovery (REC). The rate of weight gain (RW), the energy intake (EN), and the protein intake (PR) were monitored at each phase of the study. Four-hour fasting blood samples were used, and the insulin binding characteristics were investigated in the physiological range of insulin concentrations (16.7-1670 pM). Analyses of variance were used to examine differences in the variables measured at the four phases. Red cell-specific insulin binding (SB) was lower in MAL than in GI (P less than 0.001) and in (GII) (P = 0.026). SB in REC and MAL were not significantly different. Insulin receptor affinity (K) was also lower in MAL than in GI (P less than 0.001). GII (P = 0.001), and REC (P = 0.012). The insulin receptor number (S) appeared to be high in malnutrition and to decrease as recovery progressed; however the decrease was not significant. Children with fever demonstrated high insulin binding. Plasma insulin (IN) rose during recovery, and was significantly higher in GII than in MAL (P = 0.01). There was no difference in plasma glucose (G) at any phase of the study. The interrelationships among the variables measured were investigated longitudinally using multiple regression analyses, SB was positively associated with S (P = 0.032), EN (P = 0.029), and PR (P = 0.0076). S was negatively associated with K (P less than 0.001). The associations of S and K with PR were positive and approached significance (P = 0.09 and P = 0.07 respectively). RW was positively associated with PR (P less than 0.001), and with EN (P = 0.001). There were no significant relationships between G and any of the other variables longitudinally. However, correlations of the variables within phases demonstrated that in MAL, G was negatively associated with SB (P less than 0.05) and with K (P less than 0.05); but in REC, G was positively associated with SB (P less than 0.05). These results demonstrated that in severe malnutrition, the red cell insulin receptor affinity was low. During catch-up growth when protein and energy intakes were increased, both insulin receptor affinity and specific insulin binding were also increased. The negative relationship between insulin binding and plasma glucose during malnutrition may be related to carbohydrate intolerance.

Pyruvate inhibition of pyruvate dehydrogenase kinase is a physiological variable.

Pyruvate inhibited pyruvate dehydrogenase kinase activity in mitochondria from adipose tissue, heart, brain and kidney of fed rats. Starvation for 24 h led to increased kinase activity in mitochondria from adipose tissue and heart but not from brain or kidney and to reduction of pyruvate inhibition of the enzyme from adipose tissue, heart and brain. Insulin injection into starved animals rapidly restored pyruvate inhibition without alteration of kinase activity in adipose tissue and heart mitochondria. Induction of streptozotocin diabetes resulted in loss of pyruvate inhibition of the kinase in heart mitochondria at 48 h but not at 24 h whereas a significant increase of kinase activity was seen at 24 h. It is concluded that the mechanisms which control fluctuations of pyruvate sensitivity of the kinase are different from the mechanisms which control fluctuations of the uninhibited kinase activity.

Monosaccharide transport into lactating-rat mammary acini.

The uptake and release of 3-O-methyl-D-[3H]glucose at 37 degrees C by acini, prepared from lactating-rat mammary gland with collagenase, was inhibited by glucose, phloretin, cytochalasin B, HgCl2 and low temperature. Uptake and phosphorylation of 2-deoxy-D-[3H]glucose, studied in greater detail, could be ascribed to a specific, saturable, inhibitable, process of apparent Km 16 mM and Vmax. approx. 56 nmol/min per mg of protein, plus a non-specific, non-inhibitable process that was monitored with [14C]fructose. The mean rate of uptake of 5 mM-2-deoxyglucose (16 nmol/min per mg of protein) was similar to the rate of consumption of 5 mM-glucose, suggesting that transport was a rate-limiting step in the overall metabolism of glucose. This accords with evidence for a glucose gradient across the plasma membrane.

Halothane anaesthesia can block insulin stimulation of pyruvate dehydrogenase activity in mammary glands of 24-hour starved lactating rats.

The effect of halothane anaesthesia on the activity of the mitochondrial enzyme pyruvate dehydrogenase was studied in starved lactating rats. Extracts of freeze-clamped mammary gland and liver were assayed for pyruvate dehydrogenase activity. The fraction of the enzyme in the phosphorylated inactive form was increased greatly by starvation or by streptozotocin diabetes, and halothane anaesthesia did not disturb this effect. In starved animals not exposed to halothane, injection of insulin led to a rapid increase in the active fraction of the enzyme in mammary gland but not in liver. In animals under halothane anaesthesia this effect of insulin was largely abolished. The combination of starvation and halothane anaesthesia may impair mitochondrial accumulation of calcium which may be involved in the stimulation of pyruvate dehydrogenase by insulin.

The mode of regulation of pyruvate dehydrogenase of lactating rat mammary gland. Effects of starvation and insulin.

1. The ;initial activity' of the pyruvate dehydrogenase enzyme complex in whole tissue or mitochondrial extracts of lactating rat mammary glands was greatly decreased by 24 or 48h starvation of the rats. Injection of insulin and glucose into starved rats 60min before removal of the glands abolished this difference in ;initial activities'. 2. The ;total activity' of the enzyme complex in such extracts was revealed by incubation in the presence of free Mg(2+) and Ca(2+) ions (more than 10 and 0.1mm respectively) and a crude preparation of pig heart pyruvate dehydrogenase phosphatase. Starvation did not alter this ;total activity'. It is assumed that the decline in ;initial activity' of the enzyme complex derived from the glands of starved animals was due to increased phosphorylation of its alpha-subunit by intrinsic pyruvate dehydrogenase kinase. 3. Starvation led to an increase in intrinsic pyruvate dehydrogenase kinase activity in both whole tissue and mitochondrial extracts. Injection of insulin into starved animals 30min before removal of the lactating mammary glands abolished the increase in pyruvate dehydrogenase kinase activity in whole-tissue extracts. 4. Pyruvate (1mm) prevented ATP-induced inactivation of the enzyme complex in mitochondrial extracts from glands of fed animals. In similar extracts from starved animals pyruvate was ineffective. 5. Starvation led to a decline in activity of pyruvate dehydrogenase phosphatase in mitochondrial extracts, but not in whole-tissue extracts. 6. These changes in activity of the intrinsic kinase and phosphatase of the pyruvate dehydrogenase complex of lactating rat mammary gland are not explicable by current theories of regulation of the complex.