Regulation of protein synthesis by insulin.
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Animals↗
Biomedical subjects
T C Vary
Publications and source records attributed to T C Vary.
Regulation of eukaryotic initiation factor-2 expression during sepsis.
Protein synthesis is stimulated at the level of peptide chain initiation in livers from rats with a sterile or septic abscess. In contrast, peptide chain initiation is inhibited in fast-twitch skeletal muscles from septic rats. We investigated the possible mechanisms responsible for these differential changes in peptide chain initiation between liver and skeletal muscle during sepsis by measuring the cellular content of eukaryotic initiation factor-2 (eIF-2), the extent of phosphorylation of the alpha-subunit of eIF-2, and the activity of eIF-2B. In skeletal muscle, neither the eIF-2 content nor the extent of phosphorylation of eIF-2 alpha was altered during sepsis. However, a significant decrease (P < 0.001) in eIF-2B activity was observed in fast-twitch muscles. In liver, neither the extent of phosphorylation of eIF-2 alpha nor the activity of eIF-2B was different in rats with a sterile or septic abscess compared with control. However, the amount of eIF-2 in liver was increased in both sterile inflammation and sepsis. The relative abundance of eIF-2 alpha mRNA was not increased in either condition compared with control. Analysis of the distribution of eIF-2 alpha mRNA from control rats revealed that only approximately 40% of the message was associated with polysomes. Sterile inflammation or sepsis caused a 50% increase in the proportion of eIF-2 alpha mRNA associated with the polysomes compared with control.(ABSTRACT TRUNCATED AT 250 WORDS)
Actins↗
Role of elongation factor 2 in regulating peptide-chain elongation in the heart.
Cardiac muscles of experimentally induced diabetic rats show a progressive decrease in the rate of protein synthesis. The decline in protein synthesis is associated with decreases in both the number and efficiency of cardiac ribosomes. In hearts from 48 h diabetic rats, the decrease in protein synthesis was accounted for solely by a 28% reduction in the ribosome content. In contrast, the inhibition of protein synthesis in hearts from 72 h diabetic rats resulted from a reduction in both the ribosome content (28%) and the translational efficiency (30%). The decreased translational efficiency was not associated with an increase of RNA in ribosomal subunits, indicating the defect resulted from an inhibition of peptide-chain elongation/termination. Diabetes of 72 h duration resulted in a 37% inhibition in the rate of peptide-chain elongation. The decreased rate of peptide-chain elongation was associated with a 66% reduction in the amount of elongation factor 2 (EF-2). Treatment of diabetic rats with insulin for 3 days was sufficient to reverse the effects of 72 h diabetes on protein synthesis, RNA content, and translational efficiency. Also, insulin therapy increased the EF-2 content of diabetic rats to control values. These studies suggest that decreased EF-2 content is a molecular mechanism for the impaired rates of peptide-chain elongation in diabetes.
Animals↗
Analysis of physiological amino acids using dabsyl derivatization and reversed-phase liquid chromatography.
A method is described for the measurement of the specific radioactivity of primary amino acids in physiological samples. The amino acids were dabsylated followed by separation using high-performance liquid chromatography. We measured the concentration of amino acids from rat plasma or liver samples. Chromatographic analyses resolved phenylalanine from a mixture of amino acids in plasma within 30 min. An extended chromatographic gradient program completely separated all physiological amino acids within 75 min. This method is as sensitive as any current method of amino acid analysis and offers several advantages including (1) simple pre-column derivatization and (2) stability of derivatized samples.
Amino Acids↗
Insulin-induced activation of pyruvate dehydrogenase complex in skeletal muscle of diabetic rats.
The pyruvate dehydrogenase (PDH) complex undergoes reversible phosphorylation catalyzed by a PDH kinase (inactivating) and a PDH phosphatase (activating). In skeletal muscle, a decreased proportion of active PDH (PDHa) complex limits glucose oxidation in insulin-deficient states. The time-course for reactivation of the PDH complex by insulin in skeletal muscle of diabetic rats is important to understanding the potential mode of the action of insulin in regulating glucose metabolism. A single injection of insulin (1 U/kg) completely reversed the effects of alloxan-diabetes on PDHa activity within 1 hour. The normalization of the effects of diabetes on PDHa activity by insulin was maintained for a minimum of 6 hours. The increase in PDHa activity occurred before an insulin-induced decrease in plasma free fatty acids levels, demonstrating a dissociation between the antilipolytic effects of insulin and its ability to activate the PDH complex. PDH kinase activity was not normalized to control values following a single injection of insulin. Therefore, acute (1 to 6 hours) insulin-mediated activation of the PDH complex does not result from a decrease in PDH kinase activity. However, longer-term insulin therapy (1 U/kg body weight; twice daily) restored both PDHa and PDH kinase activities. The results are consistent with the hypothesis that activation of the PDH complex immediately following insulin administration is not mediated by a decreased PDH kinase activity. However, with daily insulin therapy in diabetes, activation of the PDH complex results from decreased PDH kinase activity.
Animals↗
Regulation of eukaryotic initiation factor-2B activity in muscle of diabetic rats.
Peptide-chain initiation is inhibited in fast-twitch skeletal muscle, but not heart, of diabetic rats. We have investigated mechanisms that might maintain eukaryotic initiation factor (eIF)-2B activity, preventing loss of efficiency of protein synthesis in heart of diabetic rats but not in fast-twitch skeletal muscle. There was no change in the amount or phosphorylation state of eIF-2 in skeletal or cardiac muscle during diabetes. In contrast, eIF-2B activity was decreased in fast-twitch but not slow-twitch muscle from diabetic animals. NADP+ inhibited partially purified eIF-2B in vitro, but addition of equimolar NADPH reversed the inhibition. The NADPH-to-NADP+ ratio was unchanged in fast-twitch muscle after induction of diabetes but was increased in heart of diabetic rats, suggesting that NADPH also prevents inhibition of eIF-2B in vivo. The activity of casein kinase II, which can phosphorylate and activate eIF-2B in vitro, was significantly lower in extracts of fast-twitch, but not cardiac muscle, of diabetic rats compared with controls. The results presented here demonstrate that changes in eIF-2 alpha phosphorylation are not responsible for the effect of diabetes on eIF-2B activity in fast-twitch skeletal muscle. Modulation of casein kinase II activity may be a factor in the regulation of protein synthesis in muscle during acute diabetes. The activity of eIF-2B in heart might be maintained by the increased NADPH/NADP+.
Animals↗
Carbonic anhydrase III in obese Zucker rats.
Proteins from 5- to 7-wk-old lean and obese Zucker rats were separated by one-dimensional sodium dodecyl sulfate (SDS) and two-dimensional SDS-isoelectric focusing-polyacrylamide gel electrophoresis. Laser densitometry revealed an obesity-related decrease in the concentration of a 28-kDa cytosolic adipocyte protein, the most abundant protein in adipocytes from lean Zucker rats. Microsequencing revealed the identity of this protein to be carbonic anhydrase III (CA III). The identity and obesity-related decrease was further confirmed using isoform-specific antisera and CA III enzyme activity measurements made by 18O mass spectrometry. Immunoblotting studies also revealed that CA III is present in at least two charge isoforms in adipocytes. Our data indicate that lean Zucker rat adipocytes may represent the richest source of CA III in nature (24% of the cytosolic protein content). An obesity-related decrease in both the concentration and activity of CA III was observed in two lipogenic tissues, liver and white fat, but not in soleus muscle. Adipocyte CA III activity was no longer depressed when hyperinsulinemic obese rats were made insulin deficient by streptozotocin injection. This suggests that the obesity-related decrease in CA III may be related to the hyperinsulinemia as well as to the insulin hyperresponsiveness that adipocytes from obese Zucker rats of this age display.
Adipose Tissue↗
Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts.
The myocardium responds to alterations in cardiac work by changing its rate of O2 consumption. This reflects an increase in the oxidative synthesis of ATP to meet the contractile demand for ATP. However, the biochemical mechanisms responsible for increased ATP synthesis are not fully understood. To localize the flux-controlling reaction(s) in the pathway of ATP synthesis, the effects of substrates and cardiac work on mitochondrial membrane potential (delta psi m), total tissue NADH-to-NAD+ ratio, and high-energy phosphate metabolites were examined in perfused rat hearts. Delta psi m was measured using the equilibrium distribution of tetraphenylphosphonium (33). Cytosolic phosphorylation potential, total tissue NADH-to-NAD+ ratio, and delta psi m were higher in hearts perfused with pyruvate than in those perfused with glucose. Increasing cardiac work induced a four-fold increase in O2 consumption, which was accompanied by 1) decreased or unaltered cytosolic ADP concentration, 2) increased tissue NADH-to-NAD+ ratio, and 3) decreased delta psi m. The results indicate that both NADH-generating reactions and the ATP synthase-catalyzed reaction are important in causing the increase in respiration that accompanies increased work. Because the activation of ATP synthase by cardiac work occurred in the absence of increases in delta psi m, ADP, and Pi, it is possible that the work-related acceleration in ATP synthesis may be due to modification of the kinetic properties of the ATP synthase.
Animals↗
Potentiation of decreased pyruvate dehydrogenase activity by inflammatory stimuli in sepsis.
The effect of inflammation and chronic sepsis on the activity of pyruvate dehydrogenase complex (PDH) in skeletal muscle was investigated in rats. Inflammation was induced by the placement of a catheter in the carotid artery. Sepsis was induced by repeated (every 48 hr) injections of an inoculum composed of Staphylococcus aureus, Escherichia coli, and Bacteroides fragilis organisms into a preformed subcutaneous abscess. Hindlimb muscle was sampled 7 or 14 days following the initial injection of the inoculum into the abscess. Total PHD activity was not altered by any of the conditions examined. There were no differences in the proportion of active PDH complex after 7 days in any of the conditions examined. In contrast, 14 days after the initial bacterial injection, the concentration of active PDH complex in skeletal muscle was reduced by 50% in the septic rats. The combination of intravascular catheterization and infection resulted in a further decrease in the concentration of active PDH complex. The decreased concentration of active PDH complex was associated with increased plasma lactate concentrations in septic rats. Catheterization exacerbated the rise in plasma lactate in sepsis. In this model of chronic sepsis, the magnitude of the hyperlactatemia and the inhibition of the PDH complex in skeletal muscle appear dependent upon the length of time of the septic insult and are potentiated by addition of an intravascular focus of inflammation.
Animals↗
Age-dependent decrease in the amount of eukaryotic initiation factor 2 in various rat tissues.
Recent studies have suggested that the decline in protein synthesis that occurs in rat liver and brain during development and aging is associated with a decrease in the activity of eukaryotic initiation factor 2 (eIF-2). One way in which eIF-2 activity could be decreased in tissue extracts would be through a decrease in the activity of the GDP exchange factor, eIF-2B. In the present study, the activity of eIF-2B was measured in tissue extracts and was found to be less in older than in younger rats. Thus a decrease in eIF-2B activity could account for part of the decrease in protein synthesis that occurs during aging. Another way in which eIF-2 activity could be decreased would be through a decrease in amount of the protein. Therefore the amount of eIF-2 in various tissues was quantified by protein immunoblot analysis. We found that the amount of eIF-2 relative to total protein tended to fall with increasing age. Furthermore, eIF-2 content was directly proportional to the rate of protein synthesis in the tissues examined. Finally, slot-blot analysis of polyadenylated RNA revealed no significant change in the relative abundance of eIF-2 alpha mRNA with age. The last-mentioned experiments suggest that the synthesis of eIF-2 may be regulated through changes in the deficiency of translation of eIF-2 alpha mRNA rather than through changes in gene transcription.
Aging↗
Effects of recombinant monokines on hepatic pyruvate dehydrogenase, pyruvate dehydrogenase kinase, lipogenesis de novo and plasma triacylglycerols. Abolition by prior fasting.
1. The effects of recombinant human tumour necrosis factor alpha (TNF) and murine interleukin-1 alpha (IL-1) on the activation state of the hepatic pyruvate dehydrogenase complex (PDHa), the activity of mitochondrial PDH kinase, hepatic lipogenesis de novo and plasma triacylglycerol (TG) concentrations were studied. 2. Monokine effects depended upon prior nutritional state. In rats fasted for 20 h or 45 h before monokine administration and refeeding (orally or with intravenous glucose), PDHa, TG and hepatic lipogenesis were not increased. In rats fed ad libitum, treatment with TNF plus IL-1 increased the contribution of hepatic lipogenesis to circulating TG to 550% of control values (P = 0.03) and plasma TG concentrations to 159% (P = 0.02), whereas PDHa increased slightly to 120% (P = 0.02) and liver glycogen content fell to 45.8% (P = 0.05) of control values. 3. Intrinsic hepatic PDH kinase activity was not changed by monokine treatment in rats fed ad libitum. 4. The increased lipogenesis de novo showed no correlation (r2 = 0.05, not significant) with hepatic PDHa in individual animals fed ad libitum. 5. In conclusion, these results suggest that monokines increase pyruvate flux through hepatic PDH in vivo in rats fed ad libitum primarily by mechanisms other than covalent modification of PDH. Prior nutritional status exerts a permissive effect for monokine stimulation of PDHa and lipogenesis, consistent with a substrate-mediated action, but the mechanism of this permissive effect remains uncertain.
Animals↗
Regulation of hepatic protein synthesis in chronic inflammation and sepsis.
The regulation of protein synthesis was determined in livers from control, sterile inflammatory, and septic animals. Total liver protein was increased in both sterile inflammation and sepsis. The rate of protein synthesis in vivo was measured by the incorporation of [3H]phenylalanine into liver proteins in a chronic (5 day) intra-abdominal abscess model. Both sterile inflammation and sepsis increased total hepatic protein synthesis approximately twofold. Perfused liver studies demonstrated that the increased protein synthesis rate in vivo resulted from a stimulation in the synthesis of both secreted and nonsecreted proteins. The total hepatic RNA content was increased 40% only in sterile inflammation, whereas the translational efficiency was increased twofold only in sepsis. The increase in translational efficiency was accompanied by decreases in the amount of free 40S and 60S ribosomal subunits in sepsis. Rates of peptide-chain elongation in vivo were increased 40% in both sterile inflammation and sepsis. These results demonstrate that sepsis induces changes in the regulation of hepatic protein synthesis that are independent of the general inflammatory response. In sterile inflammation, the increase in protein synthesis occurs by a combination of increased capacity and translational efficiency, while in sepsis, the mechanism responsible for accelerated protein synthesis is an increased translational efficiency.
Animals↗
Sepsis-induced changes in protein synthesis: differential effects on fast- and slow-twitch muscles.
Sepsis is associated with severe muscle wasting. Mechanisms responsible for sepsis-induced alterations in muscle protein metabolism were investigated in vivo and compared with changes induced by nonseptic inflammation. The rate of protein synthesis in mixed hindlimb muscles was not altered in inflammation but was inhibited 50% in sepsis. This inhibition did not result from a decreased RNA content. Instead, the translational efficiency was significantly reduced by 50% in skeletal muscle of septic animals compared with control. The effect of sepsis to lower the rate of protein synthesis was further examined using individual muscles containing different fiber types. Both the protein concentration and protein synthetic rate in fast-twitch muscles were reduced by sepsis, whereas neither of these parameters was affected in slow-twitch muscles or heart. The decreased translational efficiency did not result from a change in the rate of peptide-chain elongation. Instead, the sepsis-induced inhibition of protein synthesis resulted from a restraint in peptide-chain initiation because sepsis caused a 1.6-fold increase in free ribosomal subunits. Overall, sepsis, but not inflammation, caused an inhibition of protein synthesis primarily in muscles composed of fast-twitch fibers. The mechanism involved in the reduced rates of protein synthesis in muscles resulted from an inhibition of peptide-chain initiation, with no change in peptide-chain elongation.
Abscess↗
Intravascular plastic catheters. How they potentiate tumor necrosis factor release and exacerbate complications associated with sepsis.
We tested the hypothesis that long-term intravascular cannulation exacerbates the harmful effects of an infectious challenge. Four groups of rats were initially studied: rats without intravascular catheters or infection (group 1), rats without catheters with a polymicrobial infection (group 2), rats with catheters but no infection (group 3), and rats with catheters and infection (group 4). Infected animals had an increased mortality and generated a significantly increased tumor necrosis factor response compared with noninfected animals. Animals with catheters and infection generated far less cardiac output than animals from the other three groups. No histologic changes differentiated the four groups. Therefore, the presence of a sterile intravascular catheter significantly increases cardiac dysfunction and mortality rates in rats with chronic bacteremia. These results suggest that intravascular plastic catheters potentiate the destructive cascade of events produced by the host in response to bacteremia.
Abscess↗
Oxygen debt and metabolic acidemia as quantitative predictors of mortality and the severity of the ischemic insult in hemorrhagic shock.
BACKGROUND AND METHODS: An experimental canine model of hemorrhagic, hypovolemic shock is described that uses oxygen debt and its metabolic consequences of lactic acidemia and metabolic base deficit as independent variables for the prediction of probability of death. RESULTS: Lactic acidemia and metabolic base deficit are compared with the conventional hemodynamic variables of BP and cardiac output (Qt) as predictors of outcome and are shown to be superior using a modified Kaplan-Meier probability statistic. The LD50 for oxygen debt is shown to be 113.5 mL/kg, 12.9 mmol/L for lactate, and -18.8 mmol/L for base excess (BE). Comparison is made between the ability of Qt, BP, shed blood, BE, and lactate to predict oxygen debt. CONCLUSIONS: Of the single-variable predictors, BE shows the highest explained variability. However, a combined prediction from both lactate and BE appears superior to the use of either alone. Using this regression to compute the oxygen debt, it is possible to estimate accurately the actual level of oxygen debt from the BE and lactate values obtained during hemorrhagic hypovolemia. From serial determinations over time of the increase in these biochemical variables above the oxygen debt baseline, it is possible to estimate the rate of oxygen debt accumulation and the time remaining until the LD50 will be reached as indicators of the severity of the total body ischemia resulting from hemorrhagic shock.
Acid-Base Equilibrium↗
Increased pyruvate dehydrogenase kinase activity in response to sepsis.
The effect of sterile inflammation and sepsis on the proportion of active pyruvate dehydrogenase complex (PDH) in mitochondria isolated from skeletal muscle has been investigated. The proportion of active PDH in mitochondria isolated from septic animals was significantly reduced compared with control under all incubation conditions examined, even in the presence of inhibitors of the PDH kinase. There was no significant difference between control and sterile inflammation in any of the incubations examined. The rate constant for ATP-dependent inactivation of the PDH complex in mitochondrial extracts from control animals was -0.42 min-1 (r = 0.993; P less than 0.001) and was not altered in mitochondrial extracts from sterile inflammatory animals (-0.43 min-1; r = 0.999; P less than 0.001). However, rate constants for inactivation in septic animals was significantly increased over twofold to -1.08 min-1 (r = 0.987; P less than 0.001) (P less than 0.001 vs. control or sterile inflammation). In the presence of inhibitors of the PDH kinase reaction (2.5 mM pyruvate or 1 mM dichloroacetate), inactivation of PDH after addition of ATP was significantly greater in mitochondrial extracts from septic than either control or sterile inflammatory animals. These results suggest that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity.
Animals↗
Effect of dichloroacetate on plasma and hepatic amino acids in sterile inflammation and sepsis.
The effect of sterile inflammation and chronic sepsis on the plasma and hepatic free amino acid concentrations was determined. Relative to control animals, only minor alterations in the plasma amino acid concentrations were observed in sterile inflammation and sepsis. In liver, concentrations of alanine, serine, threonine, asparagine, proline, and glycine were significantly increased to the same extent in sterile inflammation and sepsis, while hepatic glutamine concentrations were significantly decreased. Compared with sterile inflammation, the branched-chain amino acid concentrations were depressed in the liver of septic animals. Following administration of dichloroacetate, hepatic alanine concentrations were significantly reduced more than threefold in each of the conditions examined; in contrast, significant increases in hepatic concentrations of threonine, glycine, glutamine, glutamate, histidine, and proline were observed. Also following administration of dichloroacetate, the branched-chain amino acid concentrations were all significantly elevated in each of the conditions examined, and plasma alanine concentrations were significantly decreased, while those of glutamine and glycine were significantly increased. These results demonstrate that there is a disassociation between the plasma and hepatic concentration of free amino acids in sterile inflammation and sepsis. Furthermore, the results demonstrate that some of the alterations in hepatic amino acid metabolism may be reversed pharmacologically by dichloroacetate.
Abscess↗
Role of anaerobic bacteria in intra-abdominal septic abscesses in mediating septic control of skeletal muscle glucose oxidation and lactic acidemia.
Altered glucose metabolism and lactic acidemia are features of Gram-negative polymicrobial abscesses, but the relationship between carbohydrate metabolism and the aerobic or anaerobic organisms is unclear. Since reductions in the % active pyruvate dehydrogenase complex (PDHa) limits glucose oxidation in sepsis, the effect of a 7-day monoclonal (E. coli or B. fragilis) vs. biclonal (E. coli + B. fragilis) intra-abdominal abscess (IA) on PDHa and lactate concentrations in skeletal muscle (SM) and plasma was studied in rats. A chronic IA was created by the intraperitoneal introduction of a sterile rat fecal-agar pellet (1.5 ml) inoculated with a known bacterial flora [sterile (S); E. coli 10(6) CFU/ml (EC); B. fragilis 10(8) CFU/ml (BF); E. coli 10(3) CFU/ml + B. fragilis 10(4) CFU/ml (ECLBF); E. coli 10(3) CFU/ml + B. fragilis 10(8) CFU/ml (ECHBF)]. Neither SM PDHa nor SM nor plasma lactate were altered from control in animals with either sterile (S) or E. coli (EC) monoclonal IA, but SM PDHa was significantly (p less than 0.001) reduced and SM lactate increased (p less than 0.05) in rats with B. fragilis 10(8)/ml (BF) monoclonal IA. In biclonal IA, the effect of sepsis on SM PDHa depended on the concentration of B. fragilis in the IA fluid (ECLBF = 10(4) CFU/ml vs. ECHBF = 10(8) CFU/ml) since the E. coli were constant (10(3) CFU/ml). At the lower B. fragilis IA concentration (ECLBF), the SM PDHa was not different from control. However, when the IA B. fragilis concentration was increased to 10(8) CFU/ml (ECHBF), the SM PDHa was significantly (p less than 0.001) decreased relative to control. A decreased muscle PDHa was always associated with elevated SM and plasma lactate concentrations. These results suggest that IA which permit a threshold of relatively nonlethal (BF = 0% mortality) anaerobic B. fragilis (greater than or equal to 10(8) CFU/ml) to enter the circulation are more important in altering metabolic control of skeletal muscle glucose oxidation and in producing lactic acidemia than are IA with only aerobic E. coli. However, in biclonal intra-abdominal abscesses, B. fragilis potentiated the early mortality from E. coli (EC = 6% vs. ECHBF = 37% mortality), suggesting that the metabolic effect of the B. fragilis-induced lactic acidemia is synergistic with the direct toxic effects of the E. coli.
Abdomen↗