Functional involvement of protein kinase C-betaII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells.

AIMS/HYPOTHESIS: Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCbetaII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements. METHODS: We examined phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCbetaII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353. RESULTS: Insulin increased phosphoPKCbetaII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCbetaII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells. CONCLUSIONS/INTERPRETATION: The results suggest that PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Protein kinase Ctheta expression is increased upon differentiation of human skeletal muscle cells: dysregulation in type 2 diabetic patients and a possible role for protein kinase Ctheta in insulin-stimulated glycogen synthase activity.

Protein kinase C (PKCtheta) is a key enzyme in regulating a variety of cellular functions, including growth and differentiation. PKCtheta is the most abundant PKC isoform expressed in skeletal muscle; however, its role in differentiation and metabolism is not clear. We examined the effect of muscle cell differentiation on PKCtheta expression in human skeletal muscle cells from normal and type 2 diabetic subjects. Low levels of PKCtheta messenger RNA (mRNA) and protein were detected in human myoblasts from both types of subjects. Upon differentiation into myotubes, PKCtheta mRNA and protein were increased 12-fold in myotubes from normal subjects. In human skeletal muscle cells obtained from type 2 diabetic subjects, increases in PKCtheta mRNA and protein were not observed upon differentiation into myotubes although expression of other markers of differentiation and fusion increased. Cells from type 2 diabetic subjects also exhibited decreased insulin-stimulated glycogen synthase activity. To determine whether the up-regulation of PKCtheta was important for the metabolic actions of insulin, PKCtheta was overexpressed in L6 rat skeletal muscle cells. Increased expression of PKCtheta occurred with differentiation of skeletal muscle myoblasts to myotubes. Glycogen synthase activity was further increased in L6 myotubes stably transfected with the complementary DNA for PKCtheta. The decreased expression of PKCtheta found in cells from type 2 diabetic subjects may be linked to insulin resistance and decreased glycogen synthase activity.

Suicide attempt by means of aspirin enema.

OBJECTIVE: To report a suicide attempt with an aspirin enema. CASE SUMMARY: A patient presented to the emergency room after self-administering, in enema form, approximately 700 aspirin tablets dissolved in water. Over the next 12 hours the patient became progressively acidemic with eventual cardiac arrest and subsequent chronic hypoxic encephalopathy. DISCUSSION: This patient's poor outcome was the result of retained aspirin products in the rectal vault combined with the failure to recognize the delayed absorption properties of rectally administered aspirin. CONCLUSIONS: In rectal aspirin overdoses, aspirin absorption from the rectum may occur over a long period of time. It is important to remove as much aspirin from the rectum as possible and to closely monitor these patients so that appropriate therapy may be started quickly. Activated charcoal given both in enema and oral form may help decrease aspirin absorption. Hemodialysis should be available and performed without delay should the patient require it.

Nitrous oxide inactivates methionine synthetase in human liver.

Activity of methionine synthetase was measured in liver biopsies of seven patients who had received 50% to 70% nitrous oxide supplemented by a combination of a narcotic and/or barbiturate with or without a volatile anesthetic, and from seven patients who were anesthetized without nitrous oxide (control group). Methionine synthetase activity (+/- SE) averaged 219 +/- 28 nmol of methionine per hour per gran of liver in patients given nitrous oxide, and 414 +/- 29 in control patients. Inactivation of methionine synthetase progressively increased as the product of the concentration of nitrous oxide and the exposure time increased. These results in humans are similar to those in animals and suggest that inactivation of methionine synthetase may play a role in the development of the pathologic effects seen in patients and medical personnel after exposure to nitrous oxide.

Uptake and metabolism of [3H]folate by normal and by vitamin B-12- and methionine-deficient rats.

The uptake of an injected dose of [3H]folic acid and its metabolism to pteroylpoly-gamma-glutamate forms by the livers and kidneys of vitamin B-12- and methionine-deficient and -supplemented rats were investigated. The initial hepatic uptake of the labeled folate dose was the same in deficient and supplemented animals, demonstrating no involvement of vitamin B-12 or methionine in folate transport. At longer time periods, a decreased hepatic net uptake of labeled folate was observed in the deficient animals compared to supplemented animals, and this was directly correlated with the decreased ability of the deficient animals to synthesize pteroylpolyglutamates. The absolute rate of loss of labeled pteroylmonoglutamate from liver was the same in deficient and supplemented animals. These data are best explained by a modification of the 'methyl trap' hypothesis for the interrelationship of vitamin B-12 and folate metabolism. Vitamin B-12 deficiency can lead to lowered levels of 5-methyltetrahydrofolate:homocysteine methyltransferase, creating a functional folate deficiency by 'trapping' an increased proportion of folate as the methyl derivative. In addition, as methyltetrahydrofolate is a poor substrate for folylpoly-gamma-glutamate synthetase, there is a decreased synthesis of pteroylpolyglutamates, the forms of the vitamin that are preferentially retained by tissues. This results in decreased tissue folate levels under conditions of vitamin B-12 deficiency. Vitamin B-12 and methionine deficiency had no significant effect on the distribution of endogenous pteroylpolyglutamates in rat liver and kidney, although total endogenous folate in rat liver was reduced by about 60%. The distribution of labeled pteroylpolyglutamates in rat liver and kidney 48 h after the tracer dose of [3H]folate closely resembled the endogenous distribution in these tissues.

Interpretation of aspiration tests in local anesthetic injections.

Three young adult laboratory rabbits were anesthetized with a barbiturate and their abdominal cavities were surgically opened to expose a variety of blood vessels. A number of veins and arteries were penetrated with 25- and 27-gauge needles attached to standard dental aspirating cartridge-type syringes. Aspiration tests were performed and the results were recorded. No differences in the performance of aspiration were detected with the two gauges of needles. Where the aperture of the needle lay unobstructed and wholly within a vein greater than 1.5 mm in diameter or within any artery, it was impossible not to aspirate blood even with a peer technique. In certain circumstances, false-negative and misleading positive readings are possible. Recommendations for a clinically reliable aspirating technique have been given and four grades of positive result described. Absolutely infallible interpretation of results is impossible but, provided the aspirating technique itself is sound, intelligent evaluation of observed effects should result in very few errors of interpretation.