Insulin modulation of pyruvate dehydrogenase in human circulating lymphocytes

1. In human circulating lymphocytes pyruvate dehydrogenase (PDH) complex is present in the active (PDHa) and inactive (PDHi) forms. 2. PDHi conversion into PDHa is stimulated when intact lymphocytes are incubated with 5 microU/ml insulin at pH 7.4, for 15 min at 37 degrees C in a medium supplemented with 50 microM Ca2+-Mg2+. 3. The generation of a mediator is strongly suggestive since a cell free preparation from circulating lymphocytes, treated as above described, still stimulates PDHi----PDHa conversion, when combined with either disrupted or intact lymphocytes.     

The insulin signal and its effects on the pyruvate dehydrogenase complex in circulating lymphocytes of obese children.

1. Studies have shown that in circulating lymphocytes pyruvate dehydrogenase (PDH) is responsive to insulin. 2. To improve existing knowledge on how insulin influences PDH behaviour, situations in which cell responsiveness to insulin is impaired could be of interest. 3. PDH behaviour in circulating lymphocytes from obese children, with high plasma insulin levels and normal glucose tolerance, was examined. 4. Masking and unmasking processes of insulin receptors on the plasma membrane appear to modulate the enzyme response to insulin.     

L-arginine stimulates insulin secretion from the pancreas of normal and diabetic rats

Summary. Several reports have shown that nitric oxide (NO) stimulates glucose-induced insulin secretion in the pancreas of normal rat but the effect of L-arginine (a NO donor) on insulin secretion from the pancreas of diabetic pancreas is unknown. Fragments of pancreatic tissue from normal and diabetic rats were incubated for 45 min in Krebs solution containing 100 mM L-arginine. The supernatant was subsequently analyzed for the insulin content using radioimmunoassay technique. L-arginine evoked large increases in insulin secretion from the pancreas of diabetic rat. The insulin secreted from the pancreas of diabetic rat was numerically but not significantly lower compared to that of normal rat pancreas. In conclusion, L-arginine, a nitric oxide donor stimulates insulin secretion from the pancreas of diabetic rats. Received October 3, 2000 Accepted November 10, 2000     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

Pyruvate dehydrogenase activation by insulin in human circulating lymphocytes and the possible pathway involved

1. The incubation of human fresh circulating lymphocytes with insulin leads to modifications in the behaviour of the pyruvate dehydrogenase complex (PDH) when the contact medium is supplemented with 50 microM Ca2+ and Mg2+. 2. To investigate the mechanism involved in the PDH responsiveness to insulin in circulating lymphocytes and the role of Ca2+ and Mg2+ in this process, the PDH activity was assayed in lymphocytes combined with insulin and/or a number of substances whose mechanism of action is partially known. 3. Of these some have been seen to mimick insulin effects on PDH, whereas other were tested for the first time in this study.     

A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats

A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats has been carried out. TheK_m for net pyruvate uptake in diabetic, ketotic mitochondria is practically equal to that measured in normal mitochondria, while theV_max is significantly lower. The lower activity of the pyruvate translocator in diabetic mitochondria compared to normal mitochondria is also shown by swelling experiments as well as by following the rate of pyruvate-supported respiration. Pre-exposure of mitochondria from normal rats to the ketone body acetoacetate and to 2-oxobutyrate results in a decrease of theK_m for pyruvate uptake. This effect is impaired in mitochondria from diabetic animals. The results indicate that the activity and the properties of the mitochondrial pyruvate translocator are modified in the diabetic, ketotic condition.Supported by a joint grant from Consiglio Nazionale delle Ricerche, Rome, Italy, and the Polish Academy of Sciences, Warsaw, Poland.     

Effect of mycoplasma infection on pyruvate dehydrogenase complex activity of normal and pyruvate dehydrogenase complex-deficient fibroblasts

The fermentative mycoplasmas A. laidlawii JS, M. hyorhinis DBS-50, M. hyorhinis GDL and M. pneumoniae FH have very high apparent activities of pyruvate dehydrogenase (PDH) (EC 1.2.4.1) and pyruvate dehydrogenase complex (PDHC). Infection of normal and PDHC-deficient fibroblasts with these mycoplasma species resulted in a marked increase of the specific activity of these two enzymes, and under certain conditions could conceal the enzymatic defect. The non-fermentative mycoplasmas M. salivarium VV and M. arthritidis PG-6 have very low apparent activities of these two enzymes. Normal fibroblasts infected with non-fermentative mycoplasmas could appear as deficient in these two enzymes. The degree of interference depends on the number of mycoplasmas associated with the harvested cells. Besides the mycoplasma species, this depends (1) on the duration of infection which determines mycoplasmal titers and also can have a killing effect on both host cells and/or mycoplasmas; (2) harvest of the cells by scraping or trypsinization; (3) centrifugal force used in the collection of the cells; (4) washing and the inherent mechanical treatment; and (5) other possibilities.     

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.     

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.     

Lactic dehydrogenase isozyme distributions in lymphocytes from normal and leukemic mice

LDH isozyme distributions were studied in the thymocytes of normal and spontaneously leukemic mice. In the normal animals, H:M ratios were found to be higher for more mature cells of adult animals than for less mature thymocytes of the neonates. However, thymocytes from leukemic animals bearing mature phenotype displayed very low values of H:M ratios. From these results, the relationship between the changes in LDH-isozyme distributions in AKR thymocytes and their progressive maturation appears to be equivocal. Alterations in isozyme distribution patterns, reflecting a decrease in H:M ratios, on the other hand, appears to be a characteristic feature of terminal stages of this murine leukemia.