Glucose Oxidation In Fat Cells Research Articles

High Fat and Highly Thermolyzed Fat Diets Promote Insulin Resistance and Increase DNA Damage in Rats

Many studies have demonstrated that DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications. The goal of this study was to evaluate the effects of the potential relationship between fat (thermolyzed) intake, glucose dyshomeostasis and DNA injury in rats. Biochemical parameters related to glucose metabolism (i.e., blood glucose levels, insulin tolerance tests, glucose tolerance tests and fat cell glucose oxidation) and general health parameters (i.e., body weight, retroperitoneal and epididymal adipose tissue) were evaluated in rats after a 12-month treatment with either a high fat or a high thermolyzed fat diet. The high fat diet (HFD) and high fat thermolyzed diet (HFTD) showed increased body weight and impaired insulin sensitivity at the studied time-points in insulin tolerance test (ITT) and glucose tolerance test (GTT). Interestingly, only animals subjected to the HFTD diet showed decreased epididymal fat cell glucose oxidation. We show which high fat diets have the capacity to reduce glycogen synthesis by direct and indirect pathways. HFTD promoted an increase in lipid peroxidation in the liver, demonstrating significant damage in lipids in relation to other groups. Blood and hippocampus DNA damage was significantly higher in animals subjected to HFDs, and the highest damage was observed in animals from the HFTD group. Striatum DNA damage was significantly higher in animals subjected to HFDs, compared with the control group. These results show a positive correlation between high fat diet, glucose dyshomeostasis, oxidative stress and DNA damage.

Studies on the effects of protease substrate analogues on some of the actions of insulin

Added TAME (N α-p-tosyl-l-anginine methyl ester) or BAME (benzoylanginine methyl ester) inhibited insulin induced activation of glucose oxidation and fat cell PDH activation without affecting spermine action on PDH activation and glucose oxidation in fat cells. BAME inhibited insulin-induced generation of both PDH stimulator and PDH inhibitor from liver particulate fraction. In contrast, insulin-induced internalization of insulin receptors and negative cooperativity of insulin receptors were not affected by protease substrate inhibitors. These results suggest that certain actions of insulin (glucose oxidation, generation of PDH regulators) are mediated by proteolytic events, while insulin-induced down regulation and negative cooperativity of insulin receptors are not mediated by activation of endogenous proteases.

The effect of selenium-deficiency on rat fat-cell glucose oxidation

When rats are fed a selenium-deficient diet, the glutathione peroxidase activity of epididymal fat-cells decreases to 5-9% of that of control rats fed the same diet supplemented with 0.5 p.p.m. of selenium as sodium selenite. [1-14C]Glucose oxidation in fat-cells from rats fed a selenium-deficient diet is unresponsive to the action of t-butyl hydroperoxide, which stimulates 14CO2 formation from [1-14C]glucose 4-fold in control rats. Insulin enhances [1-14C]glucose oxidation and incorporation into lipids in fat-cells from both groups of rats; however, the response elicited is reduced in fat-cells prepared from selenium-deficient animals. The 'C-1/C-6 ratio' (ratio of glucose C-1 to glucose C-6 oxidized) is enhanced by insulin to a similar degree in fat-cells from both groups of animals. The stimulatory action of Zn2+ and dithiothreitol on [1-14C]glucose oxidation observed in fat-cells from selenium-supplemented rats is greatly reduced in fat-cells from selenium-deficient rats. [1-14C]Glucose oxidation in fat-cells from both groups of animals is highly sensitive to the stimulatory action of adenosine. It is concluded that the enhanced formation and glutathione-linked destruction of H2O2 plays, at the most, only a minor role in the stimulation of the flux of glucose through the pentose phosphate pathway elicited by insulin, although elimination of glutathione peroxidase activity may influence the action of insulin on glucose oxidation. Production and subsequent destruction of H2O2 may play an important role in the stimulatory action of Zn2+ and dithiothreitol on fat-cell [1-14C]glucose oxidation.

Stimulation of pyruvate dehydrogenase activity in adipocytes by oxytocin: Evidence for mediation of the insulin-like effect by endogenous hydrogen peroxide independent of glucose transport

Oxytocin, a nonapeptide posterior pituitary hormone, which is known to increase glucose oxidation in fat cells like insulin, is shown here to stimulate pyruvate dehydrogenase activity in these cells. The process appears to involve the activation of preexisting molecules since there was no change in the total enzyme content after full activation. The effect of oxytocin, as well as of insulin, appears to be mediated by endogenous H 2O 2 formation, as evident from (i) the enhanced [ 14C]formate oxidation and its greater inhibition by 3-amino-1,2,4-triazole in the hormone-treated cells than in the control. This is a measure of the catalase:H 2O 2 complex, and the dose dependence of this response is found to be identical with that of glucose oxidation via the hexose monophosphate shunt pathway and of pyruvate dehydrogenase activity; and (ii) treatment of the cells with low concentration of exogenous H 2O 2 causes the activation of pyruvate dehydrogenase to the extent which is comparable with the effect of the hormones. The ED 50 of oxytocin was 7 × 10 −9 m, whereas the ED 50 of insulin was 5 × 10 −11 m. The reduced, inactive (SH) derivatives of the hormones had the same dose-response relationship, but considerably lower effect (10 to 20% of the native molecules of the hormones), indicating the significant role of the disulfide bridge(s) in eliciting these metabolic responses. The stimulation of PDH by oxytocin or insulin is found to be essentially independent of medium glucose which, however, can sustain the response apparently by recycling the intracellular oxidation-reduction state. However, unlike insulin, oxytocin fails to stimulate the rapid uptake of 3- O-[ 3H]methyl- d-glucose in these cells. The data illustrate that the major metabolic actions of insulin, viz., glucose utilization and lipogenesis, are shared by another heterologous polypeptide hormone, e.g., oxytocin, through a common effector, H 2O 2. It is suggested that (i) oxytocin may play a limited surrogate role for insulin in these cells; and (ii) H 2O 2 production may be the general basis of oxytocin's action.

Effect of amino acids on insulin-stimulated glucose metabolism in fat cells.

The effect of amino acids on insulin responsiveness in epididymal adipose tissue was examined. It was found that insulin-stimulated glucose oxidation in fat cells was significantly inhibited by glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, lysine, phenylalanine, and proline. The effect of insulin on glucose incorporation into triglyceride is also severely diminished by these amino acids. In addition, alanine reduced the incorporation of precursors ([U-14C]glucose or [1-14C]palmitate) into triglyceride both in vitro and in vivo. The Ki values of alanine were 0.4 and 0.5 mM toward the precursors of glucose and palmitate, respectively. The mechanism of reduction of insulin responsiveness in rat adipose tissue is discussed on the basis of these results.

The role of H 2O 2 formation in the insulin-like and insulin antagonistic effects on fat cells of ω-aminoalkyl glycosides

A class of ω-aminoalkyl glycosides previously found to antagonize insulin's action on glucose oxidation in fat cells and to stimulate glucose oxidation in insulin's absence is now shown to mimic insulin also on the conversion of glucose to free fatty acids and to glycerol and glycerides. These glycosides also act like insulin by inhibiting hormone- and cholera toxin-stimulated lipolysis. Various lines of evidence demonstrate that most, if not all, of the insulin-like activity of these glycosides results from H 2O 2 formed from an amine oxidase-catalyzed oxidation of the aminoalkyl moiety of these compounds. A contaminant in the bovine plasma albumin (BPA) preparations used in the bioassays was found to represent a major source of the amine oxidase activity. Membrane (ghost) preparations were also found to possess amine oxidase activity capable of forming H 2O 2 from the glycosides in amounts sufficient to express insulin-like activity. Preliminary experiments with intact adipocytes suggest that this activity is located on the cell surface. The BPA-associated activity corresponds to the known Cu 2+-containing “plasma-type” amine oxidase (EC 1.4.3.6) on the basis of its substrate specificity and susceptibility to selective inhibitors. The plasma membrane activity appears to correspond to neither the plasma-type nor to the flavin-containing mitochondrial-type (EC 1.4.3.4) and remains to be identified. The observed potent antilipolytic effects of both H 2O 2 and the aminoalkyl glycosides points out that any mechanism used to explain the insulin-like action of H 2O 2 must account for this ability to inhibit lipolysis as well as to stimulate glucose utilization. That catalase inhibits the insulin-like action of the glycosides and H 2O 2, but not that of insulin indicates that insulin's action is not mediated by cell surface-produced H 2O 2. Also, since the insulin antagonistic activity of these glycosides was not inhibited by catalase, H 2O 2 formation is not responsible for this antagonism. The latter finding, added to present and previous evidence on the carbohydrate structural requirements involved in H 2O 2 production and in the insulin-like biological and binding properties of the aminoalkyl glycosides, is consistent with a role(s) for their carbohydrate moieties in both the insulin antagonistic and agonistic activities of these compounds.

Effects of colchicine and trimethoxybenzene on glucose oxidation in isolated brown fat cells from rats

1. 1. Glucose oxidation in isolated brown fat cells from rats is reduced in the presence of colchicine. 2. 2. The effect of colchicine is seen in the absence or presence of insulin and with or without calcium in the incubation medium. 3. 3. Trimethoxybenzene is as effective as colchicine in reducing glucose oxidation in control and insulin treated fat cells. 4. 4. The ability of colchicine to reduce glucose oxidation in fat cells most likely is independent of the cytoplasmic microtubular system.

Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.

Effect of lectins on hormone release from isolated rat islets of langerhans.

Lectins from Agaricus bisporus and Agaricus campestris stimulate insulin and glucagon release from isolated rat islets in the presence of 2 mM glucose. In the case of insulin release, maximal stimulation was observed at lectin concentrations above 58 mug. per milliliter (approximately 1 muM).A. bisporus PHA-B-stimulated insulin release was independent of a source of metabolic energy but was abolished by deuterium oxide. The lectin did not alter islet glucose oxidation to CO2 or incorporation of [3H] leucine into trichloracetic acid-precipitable material nor did it modify rates of insulin secretion induced by 20 mM glucose. None of nine other lectins tested stimulated insulin release, whereas stimulation of fat cell glucose oxidation was a general property of the lectins. Binding of 125I-labeled A. bisporus PHA-B to islets increased with time up to one hour and after attainment of equilibrium was very slowly reversible. Binding was directly proportional to islet number and the estimated Kdiss of the binding reaction was 17 mug per milliliter. The total number of A. bisporus PHA-B binding sites per islet was approximately 2 times 10(10). Binding of A. bisporus PHA-B to the islets and A. bisporus PHA-B-stimulated insulin release were inhibited in parallel by a glycopeptide containing the oligosaccharide receptor for the lectin, suggesting that lectin binding is essential for the expression of insulin-releasing activity. It is proposed that the specific interaction between mushroom lectin and its receptors may lead to conformational changes in the structure of the membranes of the islet A2- and B-cells that facilitate exocytosis.

Effects of bovine growth hormone preparations, fragments of growth hormone and pituitary anti-insulin peptide on lipolysis and glucose metabolism of isolated fat cells and adipose tissue.

The effects on rat fat cells and adipose tissue of a bovine adenohypophysial anti-insulin peptide (AIP) were compared to those of various bovine growth hormone preparations. The lipolytic activity of AIP was about 1–2% that of growth hormone on a weight basis. The increase in lipolysis seen with AIP was blocked by cycloheximide. AIP did not alter basal glucose oxidation in fat cells but slightly increased basal glucose oxidation in adipose tissue. AIP also did not inhibit the insulin stimulation of glucose oxidation in free fat cells or adipose tissue. Growth hormone inhibited the increase in glucose oxidation due to insulin but this effect was dependent on lipolysis. Dexamethasone markedly inhibited insulin-stimulated glucose oxidation despite its failure to activate lipolysis. In the presence of dexamethasone the increase in lipolysis due to growth hormone was enhanced. The lipolytic activity of highly purified bovine growth hormone preparations was greater than that of the NIH preparation. The fragment...

Evidence for Electron Transfer Reactions Involved in the Cu2+-dependent Thiol Activation of Fat Cell Glucose Utilization

Abstract Previous studies indicated that the ability of thiols to mimic insulin action on fat cell glucose oxidation and lipolysis was dependent on Cu2+. Evidence now presented indicates that H2O2 formed by the reaction of thiols, Cu2+, and O2 mediates the effects of these agents on isolated fat cells. Oxidation of thiols to the respective disulfides in the presence of Cu2+ accompanied the stimulatory effects on glucose metabolism. Diphenyl-1, 10-phenanthroline, which binds tightly to Cu+ but not Cu2+, blocks copper-catalyzed thiol oxidation and the stimulatory effect on fat cell glucose utilization, indicating that reduction of Cu2+ is an obligatory step for thiol action. The thiol-Cu2+ effect on fat cells is readily reversible since stimulated rates of glucose oxidation returned to control levels following addition of the chelator. Catalase inhibited the stimulatory effect of thiols but not insulin on fat cell glucose oxidation, whereas H2O2 mimicked the action of thiols and insulin on this process. As little as 10 µm H2O2 stimulated glucose utilization, whereas 1 mm H2O2 was maximally effective. Higher concentrations were less effective. The stimulatory effect of various mercaptoethanol and Cu2+ concentrations on labeled CO2 production from d-[1-14C]glucose by fat cells paralleled the peroxide formed under these conditions. Other oxidants such as MnO4= and diamide were also effective in enhancing fat cell glucose oxidation. The stimulatory effect of all three oxidants, as well as insulin, was inhibited by phlorizin and cytochalasin B, known glucose transport inhibitors. Fat cells enhanced the decomposition of added H2O2 or that generated by cysteine, Cu2+, and O2. These data are consistent with the concept that an electron transfer reaction between a fat cell component or components and oxidants results in stimulated glucose transport or metabolism or both.

Stimulation of glucose metabolism by lectins in isolated white fat cells

Addition of 5 μg/ml concanavalin A to isolated white fat cells in the presence of 1 % albumin maximally stimulated the conversion of d-[1- 14C]glucose to CO 2, glyceride-glycerol and fatty acids over a 1 h incubation period; as little as 1 μg/ml agglutinin increased fat cell glucose oxidation more than 2-fold. Labelled CO 2 production in the presence of concanavalin A was linear for at least 90 min and was inhibited by 40 mM α-methyl- d-glucoside which had little effect on basal or insulin-stimulated glucose oxidation. The effect of a submaximal concentration of the agglutinin was additive to that of submaximal but not maximal concentrations of insulin. Concanavalin A caused agglutination of fat cells which could be readily detected by light microscopy. Digestion of fat cells with 0.5 mg/ml trypsin for 15 min did not affect subsequent agglutination and inhibited the increased glucose oxidation due to concanavalin A by less than 30%. Thus the action of concanavalin A was much less sensitive to trypsinization of fat cells than insulin since trypsin under the above conditions completely abolished the effect of insulin. An anti-blood group A agglutinin from Phaseolus lunatus and Lens culanaris agglutinin also markedly stimulatedfat cell glucose conversion to CO 2. Agglutinin-stimulated glucose metabolism was inhibited by phloretin. This binding of several types of specific plant lectins to fat cell membrane glycoprotein(s) and/or glycolipid(s) apparently initiates events which results in increased glucose transport.

Cu++-dependent Thiol Stimulation of Glucose Metabolism in White Fat Cells

Abstract EDTA, added either alone or with equimolar concentrations of CaCl2 or MgCl2, was without effect on the increase in fat cell glucose oxidation due to insulin, vitamin K5, menadione, or sucrose hyperosmolarity. In contrast, EDTA markedly inhibited the action of cysteine, glutathione, and an insulin-like factor from serum on fat cell glucose metabolism. The inhibition by EDTA was greater on high concentrations of cysteine (1.5 or 2 mm) than lower concentrations (0.5 or 1 mm). In fat cells incubated in a 3% albumin medium which had been incubated for 30 min with 2 mm cysteine prior to addition of EDTA and cells, EDTA was ineffective in blocking the action of cysteine. The stimulation by cysteine, glutathione, and vitamin K5 of white fat cell glucose oxidation was inhibited in the absence of albumin while the response to insulin or sucrose hyperosmolarity was unaffected. The effect of albumin on the action of cysteine was biphasic. The stimulatory effect of 0.05 or 0.5 mm cysteine was about 3-fold greater than 1.5 mm cysteine in the presence of 0.75% albumin while with 3% albumin present the response of cells to 1.5 and 0.5 mm cysteine was maximal and that to 0.05 mm cysteine was nearly abolished. Dialysis of albumin reduced its ability to support the effect of cysteine as did passage over Sephadex G-50. The stimulation by cysteine and glutathione of glucose metabolism was inhibited by almost 90% if fat cells were incubated in albumin which had been exposed to 3.3 mm phenanthroline and then chromatographed on Sephadex G-50 while the increase due to insulin was diminished by only 25%. Cu++ at 3 x 10-6 and 10-5 m, but not 10-4 m, restored the stimulatory effect of cysteine on fat cell glucose metabolism in the presence of the phenanthroline, Sephadex-treated albumin. These results indicate that the stimulatory effects of cysteine on white fat cell glucose utilization involve a mechanism dependent on the presence of divalent copper.

Antagonism of insulin action on glucose metabolism in white fat cells by dexamethasone.

Treatment of white fat cells with dexamethasone during a 1–2–hr isolation period inhibited the stimulatory effect of insulin on [l–14C]—D—glucose conversion to CO2 during a subsequent 1 hour incubation under the following two conditions: 1) Submaximal doses of insulin in the presence of 1 HIM glucose, and 2) A maximal dose of insulin and 0.05 mM or 0.1 mM glucose. Under conditions of high rates of labeled CO2 production which occur with a maximal dose of insulin and 1 mM or more glucose, dexamethasone was without effect on the response of white fat cell glucose oxidation to insulin. Isolation of fat cells in the presence of 8.1 X 10-8M dexamethasone followed by incubation with the glucocorticoid resulted in marked inhibition of the insulin—like effects of 1 mM cysteine and 2.8 X 10-7M prostaglandin E1 in the presence of 0.1 mM glucose. Cycloheximide mimicked the action of dexamethasone on labeled CO2 production in the absence or presence of insulin or cysteine when fat cells were incubated with 0.05 mM gl...

Studies with Tritiated Polypeptide Hormones: II. BIOLOGICAL AND CHEMICAL PROPERTIES OF TRITIATED ACETAMIDINO-INSULIN

Abstract Tritiated acetamidino derivatives of insulin are readily prepared. That the reaction is specific for amino groups has been confirmed and the mildest conditions for optimal labeling established. At acid pH, there is no significant loss of tritium from the polypeptide. A small loss of 3H occurs after 3 months at pH 9. Acetamidino-insulin is fully potent in the following assays: mouse convulsion, muscle sugar transport, fat cell glucose oxidation, and immunoassay. A slight loss of potency was observed in some cases with high specific activity preparations. Acetamidination introduces some heterogeneity into insulin preparations, but the products appear to be biologically active and can be separated by disc gel electrophoresis.