Search

Oxidative stress has been implicated in glucose toxicity. We tested the hypothesis that certain antioxidants may prevent insulin-resistant glucose transport that develops in adipocytes after sustained exposure to high glucose, provided insulin is present. The antioxidant alpha-lipoic acid has been proposed as an insulin sensitizer. 3T3-L1 adipocytes were preincubated 18 hours in media containing insulin (0.6 nmol/L) with low (5 mmol/L) or high (25 mmol/L) glucose with or without alpha-lipoate, dihydrolipoate (each 0.1 to 0.5 mmol/L), or N-acetylcysteine (1 to 5 mmol/L). After extensive re-equilibration in insulin and antioxidant-free media, basal and maximally insulin-stimulated (100 nmol/L) glucose transport was measured. Insulin was quantified by radioimmunoassay. Preincubation with alpha-lipoate and dihydrolipoate but not N-acetylcysteine increased subsequent basal glucose transport; the effect was much smaller than that of acute maximal insulin stimulation. Preincubation in high glucose without antioxidants inhibited acutely insulin-stimulated glucose transport by 40% to 50% compared with low glucose. This down- regulation was partially or completely prevented by each antioxidant. In cell-free media, the 2 reductants, dihydrolipoate and N-acetylcysteine, rapidly decreased immunoreactive insulin, but alpha-lipoate was ineffective. However, during incubation with adipocytes, alpha-lipoate, and dihydrolipoate promoted the decline in immunoreactive insulin nearly equally. Because insulin and high glucose are synergistic in inducing insulin resistance in this model, the reduction in immunoreactive insulin probably contributed to the protective effect of the antioxidants. 3T3-L1 adipocytes efficiently metabolize alpha-lipoate to dihydrolipoate, which may be released into the medium. The stimulation of glucose transport by alpha-lipoic acid may represent redox effects in subcellular compartments that are accessible to dihydrolipoate., (Copyright 2001 by W.B. Saunders Company)

Insulin treatment increases the SN-1,2-diacylglycerol (DAG) concentration in skeletal muscle. Because DAG may participate in transmission or modulation of the insulin receptor signal, we examined the effect of insulin on total DAG and on different DAG species in isolated rat hemidiaphragms incubated with 5 mmol/L glucose. Five DAG species (16:0-18:1 omega 9, 16:0-18:1 omega 7, 18:0-18:1 omega 9, 18:0-18:2 omega 6, and 18:1-18:2) were identified and quantified. After a 5-minute incubation with 60 nmol/L insulin, neither total DAG nor a DAG species increased; exposure to insulin for 10 or 20 minutes increased the concentration of total DAG and of several DAG species. Insulin did not increase DAG in muscles incubated without glucose. Two sources for the insulin-mediated DAG increase were considered: phosphatidylcholine (PC) hydrolysis and de novo DAG synthesis from glucose. Concentrations of choline and phosphocholine in muscle were not increased after 10-minute incubations with insulin. However, insulin increased 14C incorporation from [U-14C]glucose into DAG, triacylglycerol (TAG), and total lipids approximately threefold. Okadaic acid (OKA), an inhibitor of phosphoprotein phosphatases 1 and 2A, increased muscle DAG content and synthesis from glucose, similar to the effect of insulin. Doses of OKA or insulin that increased DAG mass greatly exceeded those required for stimulation of glucose transport. The insulin-mediated, relatively slow increase in muscle DAG observed here likely reflects primarily de novo synthesis from glucose. This effect would be downstream of insulin stimulation of glucose transport. However, a possible insulin-mediated, rapid transient increase in muscle DAG content and PC hydrolysis cannot be ruled out by our studies.

Three days after denervation, the expression of GLUT4 mRNA and protein decreases by approximately 50% in rat hindlimb muscles, while GLUT1 mRNA increases transiently by approximately 500%. Although postreceptor insulin resistance of glucose transport develops before GLUT4 declines, the latter likely contributes to the time-dependent increased severity of the resistance. To determine whether muscle inactivity contributes to changes in glucose transporter expression, one rat hindlimb was immobilized in a plaster cast for 3 days; the unencumbered hindlimb served as control. Muscle GLUT4 mRNA decreased by 32% (P < .02) and GLUT4 protein by 40% (P < .05) after 3 days' immobilization. There was no significant change in GLUT1 mRNA or skeletal muscle alpha-actin mRNA expression or in the total RNA concentration. The data suggest that electromyogenic and/or contractile activity regulates GLUT4 expression in skeletal muscle at a pretranslational step.

The free amino acid content of diaphragm muscles of control and diabetic rats was studied 5 days after the injection of streptozotocin. Muscles were prepared for analysis either immediately after sacrifice or following incubation in balanced salt solution containing 5.5 mM glucose, with or without an electron acceptor, 0.02 mM methylene blue. Diaphragms of diabetic rats contained significantly more free taurine, glutamate, and branched chain amino acids than the controls at sacrifice, and significantly less glutamine, serine, asparagine, lysine, arginine, histidine, threonine, citrulline, and carnosine. Alanine decreased in plasma of diabetic rats but not in diaphragms before incubation. Hemidiaphragms of diabetic rats produced less alanine and more glutamate during incubation than controls. After incubation they contained less than half as much alanine and glutamine and twice as much glutamate than the controls, having released approximately 40% less alanine and 25% more glutamate into the medium than the controls. Glutamine release was not significantly different between the two groups. Methylene blue increased the free alanine content in the tissue water as well as alanine release by control and by diabetic muscles; the glutamate content of muscles decreased concomitantly. The effects of methylene blue were greater in the diabetic group. Branched chain amino acid release by diabetic muscles decreased during incubation with methylene blue. Muscles of diabetic rats contained more alpha-ketoglutarate than the controls after incubation with or without methylene blue. Methylene blue increased the alpha-ketoglutarate content of muscles and its release into the medium, the effect being greater in diabetics than in controls. Hemidiaphragms from diabetic rats released less pyruvate during incubation than controls, while lactate release by the two groups was not significantly different. Incubation with methylene blue caused a marked increase in pyruvate release by diabetic muscles, and a lesser stimulation in controls; lactate release increased in both groups. After incubation the lactate/pyruvate ratio in muscles was lower in the methylene blue treated group. The in vitro effect of 0.02 mM phenazine methosulfate on alanine production was similar to that of methylene blue. The data is compatible with the hypothesis that the NADH/NAD ratio may exert a restraining effect on alanine production and release by muscle. The progressive increase in this ratio may play a role in the eventual deceleration of gluconeogenesis during a prolonged fast and may restrain this process in uncompensated diabetes.

The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, increases after a protein meal. The effect of amino acid mixtures on hepatic and renal ODC activity and polyamine content was studied in postabsorptive and 72-hour fasted rats. Fasting decreased ODC activity in liver and in kidney by approximately 50%. Hepatic ODC activity increased tenfold 4 hours after intraperitoneal injection of either 1 g/kg of a synthetic mixture of 17 amino acids or of casein hydrolysate to fed rats and about 20-fold in fasted rats. Renal ODC activity increased four- and tenfold respectively. A mixture of glutamate, aspartate, and alanine at concentrations given in the hydrolysate reproduced the full amino acid effect. No amino acid was effective when given alone, nor were mixtures of the other amino acid constituents of the hydrolysate. Glutamate + alanine was ineffective as were glucose or various combinations of arginine, ornithine, aspartate and NH3. Ornithine + glutamate or aspartate + glutamate were active but stimulated less than aspartate + glutamate + alanine. Hepatic and renal putrescine content increased in parallel with ODC activity. The data suggest that specific amino acids possess the full ODC-stimulating capability of a high quality protein and that polyamine synthesis is linked to urea cycle activity.

In vitro catabolism of branched-chain amino acids, leucine and valine, was investigated using diaphragm muscles from normal, streptozotocin-diabetic and overnight fasted rats. Oxidation and transamination of [1-14C] branched-chain amino acids were both stimulated to a similar extent by diabetes or fasting, when diaphragms were incubated with glucose. Transamination of leucine and valine was increased when diaphragms were incubated with pyruvate; stimulation of transamination was greatest in diaphragms from diabetic rats. Leucine and valine oxidation by control diaphragms was inhibited by pyruvate while it was unchanged or slightly stimulated in diaphragms from fasted or diabetic rats. Thus diaphragms from diabetic rats oxidized two to threefold more branched-chain amino acids than controls when they were incubated with pyruvate. The specific radioactivity of extracellular alpha-ketoisocaproate (KIC; the product of leucine transamination) produced by diaphragms incubated with [14C]leucine was similar for all groups (fed, fasted, or diabetic) in the presence or absence of pyruvate. Oxidation of [1-14C]KIC by diaphragms from fasted or diabetic rats, incubated with glucose, was the same or less than KIC oxidation by control diaphragms. Incubation with pyruvate inhibited KIC oxidation by control diaphragms to a significantly greater degree than that by diaphragms from diabetic or fasted rats. These data suggest the following Flux through branched-chain amino acid transaminase is limited by the availability of amino group acceptors in diaphragms from normal and overnight fasted rats, and to a greater extent in diaphragms from diabetic rats. Flux through the transaminase may be a major determinant of accelerated branched-chain amino acid oxidation by diaphragms in fasting and diabetes. In diaphragms of fasted and diabetic rats, flux through the branched-chain alpha-ketoacid dehydrogenase complex is resistant to inhibition by pyruvate, which is normally observed in controls.