Glucose Oxidation In Rat Adipocytes Research Articles

The effects of polybrominated diphenyl ethers (PBDEs) on adipocyte functionality in vitro and in vivo

PBDEs are flame retardants found in furniture, carpets and electronics. These organic pollutants are highly lipophilic and accumulate in the adipose tissue of animals and humans. However, the effects of PBDEs on adipocyte metabolism are unknown. The purpose of this study was to determine the effects of penta-PBDE on lipolysis and glucose oxidation in rat adipocytes in vitro and in vivo. In vitro, adipocytes from 12 male Sprague-Dawley rats were isolated and incubated for 90 minutes with either 0, 15 or 30ug/ml PBDEs (4 rats/group.) In vivo, male Sprague-Dawley rats weighing 120g were gavaged daily for 4 weeks with either 18mg/kg PBDEs or corn oil (12 rats/group). Adipocytes from in vivo and in vitro treatments were tested for lipolytic response to epinephrine and [U-14C] glucose oxidation response to insulin. Rat weight gain was similar in all groups throughout the study. In vitro data show that incubation with PBDEs for 90 minutes had no effect on lipolysis or glucose oxidation in rat adipocytes. In vivo data show that after 4 weeks, basal lipolysis was increased by 58% and maximal lipolysis was increased by 22%, compared to control. There was a 41% decrease in maximal glucose oxidation in rats treated with PBDEs for 4 weeks, compared to controls. In summary, although incubation of adipocytes with PBDEs does not change adipocyte functionality, a low daily dose of PBDEs to rats increased basal and maximal lipolysis and decreased maximal glucose oxidation in freshly isolated rat adipocytes. Whether these changes persist and contribute to permanently altered adipocyte function remains to be determined. Supported by Agr. Exp. Stn. Grant NH00432.

Isolation and characterization of two different insulins from an amphibian, Xenopus laevis.

The South African clawed toad, Xenopus laevis, is a versatile laboratory model of vertebrate development. To study the role of insulin during embryogenesis, we have recently cloned preproinsulin cDNAs from this species. Unexpectedly, we identified two preproinsulin cDNAs corresponding to two different nonallelic genes that code for similar but distinctly different insulins. We now report the isolation, amino acid sequence, and characterization of both of these insulins from pancreatic extracts of adult toads, confirming that both Xenopus preproinsulin genes are expressed. Xenopus insulins represent the first amphibian insulins to be characterized. Xenopus insulin I and Xenopus insulin II are more similar to each other than they are to insulins of other species. In addition, Xenopus insulins are more similar to mammalian and bird insulins, than they are to fish insulins, implying a closer evolutionary link to terrestrial vertebrates than to most aquatic vertebrates. A homogeneous preparation of Xenopus insulin I showed high reactivity in a pork insulin RIA. Xenopus insulin I was approximately 2-fold more potent than pork insulin in binding to insulin receptors on human IM-9 lymphocytes and 1.5-fold more potent than pork insulin in stimulating glucose oxidation in rat adipocytes. We were unable to purify Xenopus insulin II sufficiently for immunological and biological characterization.

Insulin-stimulated Hexose Transport and Glucose Oxidation in rat Adipocytes Is Inhibited by Sphingosine at a Step after Insulin Binding

Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin.

Autoantibodies to the Insulin Receptor (B-10) Can Stimulate Tyrosine Phosphorylation of theβ-Subunit of the Insulin Receptor and a 185,000 Molecular Weight Protein in Rat Hepatoma Cells*

The immunoglobulin G (IgG) fraction obtained from the serum of a patient (B-10) with type B insulin resistance and acanthosis nigricans stimulated both glucose oxidation in rat adipocytes and autophosphorylation of tyrosine residues in the beta-subunit of insulin receptors in H-35 hepatoma cells. Partially purified insulin receptor from H-35 cells, when incubated with B-10 IgG, had increased tyrosine kinase activity for a synthetic peptide sequentially similar to the site of tyrosine phosphorylation in pp60v-arc (the gene product responsible for cellular transformation by the Rous sarcoma virus). In H-35 cells, both B-10 IgG and insulin stimulated tyrosine phosphorylation in an endogenous 185,000 mol wt protein. This phosphoprotein may be similar to the cellular substrate for insulin in hepatoma and other cultured cell lines demonstrated by others. These results suggest that antiinsulin receptor antibodies (B-10) may initiate their insulin-like effects via tyrosine phosphorylation of the insulin receptor, activation of its tyrosine kinase activity, and phosphorylation of a cellular protein substrate of 185,000 mol wt.

Anti-insulin receptor antibodies in human diabetes

Sera and immunoglobulin G from 10 104 diabetic patients (five with insulin-dependent, five with non-insulin-dependent diabetes) were found to contain antibodies that bound 125I-labelled purified human placental insulin receptors. Nine of these sera failed to inhibit insulin binding to cultured rat hepatocytes and did not stimulate glucose oxidation in rat adipocytes. Only one serum modestly inhibited insulin binding and stimulated glucose oxidation. These results suggest that sera from nine of these 104 diabetics contained a new type of anti-insulin receptor antibodies (AIRA) which bound to a locus different from the insulin binding site, and that only one of the 104 diabetic sera contained a low titer of conventional AIRA which could cause a clinical condition not distinguishable from ordinary non-insulin-dependent diabetes.

Preparation and characterization of a colloidal gold-insulin complex with binding and biological activities identical to native insulin.

We studied the binding and biological activities of gold-insulin complexes to develop a complex with properties identical to native insulin. Stabilizing amounts of insulin absorbed to 5-, 10-, or 15-nm gold particles resulted in complexes with 40-327 insulin molecules per gold particle and 4-111 times the biological activity of unlabeled insulin, based on the molar concentration of gold complex. These data suggested that these complexes behaved as multivalent ligands. Gold-insulin complexes were prepared with 5% of the stabilizing insulin concentration and were stabilized with bovine serum albumin. This resulted in a complex with 5-7 insulin molecules per 10-nm gold particle, which stimulated glucose oxidation in rat adipocytes and competed with [125I]-insulin for binding to the insulin receptor identically to unlabeled insulin on an equimolar basis. The organization and distribution of insulin receptors occupied by this monovalent-behaving gold-insulin complex were virtually identical to previous observations using monomeric ferritin-insulin. Since multivalent ligands may affect receptor binding, re-distribution, and intracellular processing, the use of electron-dense probes that resemble the unlabeled ligand in biological and binding properties is appropriate when studying receptor dynamics of in vivo or in vitro biological systems. The gold-insulin complex developed in this study should serve this function.

Structurally abnormal insulin in a diabetic patient. Characterization of the mutant insulin A3 (Val----Leu) isolated from the pancreas.

We have recently identified a diabetic patient with marked fasting hyperinsulinemia. Family study revealed that the abnormality was an autosomal dominant trait. High-performance liquid chromatography (HPLC) profile of the patient's serum insulin showed that she had an abnormal insulin in addition to a normal insulin. We have purified her insulin(s) from the specimen of her pancreas, which was biopsied during an operation of cholelithiasis. Insulin was also immunologically purified from the serum of her portal vein. The reverse-phase HPLC analysis revealed that the ratios of normal to abnormal insulin in the pancreas, portal vein, and peripheral vein were 5:4, 4:5, and 1:7, respectively. Radioreceptor assay for insulin using guinea pig kidney membrane revealed that the binding activities of the normal component insulin, the abnormal component insulin and her pancreatic insulin containing both components were 100, 5, and 50% of standard human insulin, respectively. The biological activities of the normal component, the abnormal component and her pancreatic insulin to stimulate glucose oxidation in rat adipocytes were found to be 100, 8, and 60% of standard human insulin, respectively. Analysis of amino acid sequences of the abnormal insulin purified from her pancreas strongly suggested the substitution of leucine for valine at the third position of the A chain, A3 (Val----Leu).

Insulin like action of oxytocin

The insulin-like activity of oxytocin in stimulating glucose oxidation in rat adipocytes has been demonstrated repeatedly in the last 20 years. Oxytocin binds to a specific cell surface receptor of adipocytes; however, little attention has yet been paid to the effect of oxytocin on glucose oxidation in other tissues. We have initiated studies into the metabolicregulatory activity of oxytocin in insulin sensitive tissues and uterus.

Effect of N6-methyladenosine on fat-cell glucose metabolism : Evidence for two modes of action

N 6-Methyladenosine strongly stimulates [1- 14C]glucose oxidation in rat adipocytes [J.E. Souness and V. Chagoya de Sanchez, Fedn Eur. Biochem. Soc. Lett. 125, 249 (1981)]. The effect of the adenosine analogue is largely independent of its action as an R-site agonist. Removal of endogenous adenosine was a prerequisite for the manifestation of the effect of N 6-methyladenosine. Nucleoside uptake inhibitors, dipridamole and nitrobenzylthioinosine, did not block the action of N 6-methyladenosine on [1- 14C]glucose oxidation. The effect of the adenosine analogue was not greatly influenced by N 6-phenylisopropyladenosine, nicotinic acid or theophylline. Although N 6-methyladenosine stimulated 3- O-methylglucose uptake into fat cells, it is uncertain whether this was its only effect on glucose metabolism, in view of the comparable enhancement of hexose transport elicited by N 6-phenylisopropyladenosine, a much weaker stimulator of glucose oxidation. That hexose transport is not the sole site of action of N 6-methyladenosine was supported by the finding that, under conditions which have been proposed to make glucose transport rate limiting, the adenosine analogue only weakly enhanced [1- 14C]glucose oxidation. The conversion of glucose carbon 1 to 14CO 2 was enhanced by N 6-methyladenosine to a greater degree than that of carbon 6, suggesting an increase in pentose phosphate shunt activity. Mechanisms by which this could arise are discussed. Although similarities exist between the effects of insulin and N 6-methyladenosine on adipocyte glucose metabolism, the mechanisms by which they stimulate glucose oxidation appear to be distinct, in view of the additivity of their actions on [1- 14C]glucose conversion to 14CO 2. The results indicate that N 6-methyladenosine affects fat-cell glucose metabolism via two different mechanisms: (1) a weak R-site-dependent mode of action related to stimulation of glucose transport and inhibition of lipolysis, and (2) a strong R-site-independent effect of unknown mechanism.

Effects of prednisolone and dexamethasone in vivo and in vitro: studies of insulin binding, deoxyglucose uptake and glucose oxidation in rat adipocytes.

We have studied the effects of dexamethasone and prednisolone in vitro and in vivo on insulin binding, deoxyglucose uptake and glucose oxidation in rat adipocytes. In the studies in vivo, rats were treated for 22 h with dexamethasone (30 micrograms/kg) or prednisolone (200 micrograms/kg). Following sacrifice, adipocytes were prepared and the results demonstrated that cells from prednisolone treated rats showed a 17% increase in insulin binding and increased rates of basal and insulin stimulated deoxyglucose uptake and glucose oxidation. Conversely, dexamethasone administration resulted in a 22% decrease in insulin binding, and decreased rates of deoxyglucose uptake and glucose oxidation by the cells. Thus, prednisolone and dexamethasone had opposite effects in vivo. In contrast to the opposite effects of the two glucocorticoids in vivo, dexamethasone and prednisolone (each at a concentration of 1 mumol/l) had similar effects on adipocytes in vitro. Incubation of adipocytes with the steroids did not alter insulin binding, while both agents led to a comparable decrease in the rates of basal and insulin stimulated deoxyglucose uptake and glucose oxidation. Thus, dexamethasone and prednisolone have opposite effects on adipocyte glucose metabolism in vivo but have similar effects in vitro.

Changes in prostaglandin E 1 stimulation of glucose oxidation in rat adipocytes during maturation and aging

Prostaglandin E 1 stimulates glucose oxidation in isolated rat adipocytes in a time and concentration dependent manner. Maximal stimulation requires 2 hours exposure to prostaglandin, although effects can be detected by 0.5 hours or earlier. In contrast to prostaglandin E 1, prostaglandin F 2 α has essentially no effect on glucose oxidation. Maximal stimulation by prostaglandin E 1 at all ages tested occurs at concentrations of 10 −5 − 10 −4M. Stimulation is greatest in cells of mature (10–12 month old) animals at 81 ± 9% above basal levels of glucose oxidation. This is to reduced to 48 ± 8% in cells of senescent (23–26 month old) animals, and at 23 ± 18% in cells of young (2–3 month old) rats is not significantly different from basal oxidation in most animals. These results are consistent with data for adipocytes and other cell types indicating that responsiveness to certain hormones is altered during maturation and aging.

Changes in Hormone Action during Aging: Glucocorticoid Regulation of Adipocyte Glucose Metabolism and Catecholamine Regulation of Myocardial Contractility

AbstractMany responses to hormones are altered during aging. Almost all the cellular components involved in mediating hormone actions have, in one situation or another, been implicated in such responsiveness changes. Glucocorticoid inhibition of glucose oxidation in rat adipocytes and catecholamine stimulation of rat heart contractile performance are two examples of reduced hormonal responsiveness during aging. In the former system, age changes have been reported in glucocorticoid receptor concentrations, rates of putative receptor biosynthesis, regulation of the glucose transport system, and the sulfhydryl content of the plasma membrane. In the latter system, no changes have been observed in catecholamine receptors, cyclic AMP production, or protein kinase activation. Instead, the age-associated functional change appears to be related to control of calcium entry into the cell. The manifestations of aging at the level of hormone action, therefore, appear to be multifaceted.

Insulin-like stimulation of glucose oxidation in rat adipocytes by vanadyl (IV) ions

The mechanism of insulin action is still unknown1. One approach to this problem is to apply substances which mimic the action of the hormone to target cells. Ouabain and deprivation of extracellular K+ (refs 2,3), which inhibit the active transport of Na+ and K+ ions, are both known to activate glucose transport and oxidation in isolated adipocytes. Vanadate (V) ions have recently been shown to act as very efficient inhibitors of the sodium pump or (Na+ + K+)ATPase in in vitro preparations4. They have a natriuretic and diuretic effect in rats5 and a positive inotropic effect on cat heart muscle6. Many tissues contain vanadium at a concentration of about 0.1–1.0 μM (ref. 7) and so endogenous vanadate could be a physiological regulator of the sodium pump. But this is still open to debate, because the bulk of the vanadium is probably in the vanadyl (IV) form8,9 and VO2+ ions bind tightly to proteins8,10. VO2+ is a relatively ineffective inhibitor of (Na+ + K+) ATPase in vitro8,11. We report here that externally applied vanadate ions at low concentrations mimic fully the effect of insulin on glucose oxidation in rat adipocytes. However, this simulation seems to be due mainly to the effects of vandyl (IV) ions, probably produced within the cells, and not primarily to inhibition of the sodium pump. Also, externally applied vanadyl (IV) ions stimulate glucose oxidation substantially. Vanadyl ions are known to be powerful inhibitors of alkaline phosphatase12 and we therefore consider the possibility that they inhibit a cellular phosphatase activity. An early event in insulin action may involve alteration of the degree of phosphorylation of protein(s) involved in regulation of sugar transport.

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.

Comparative study of the effects of porcine proinsulin and insulin on lipolysis and glucose oxidation in rat adipocytes.

Proinsulin blocked lipolysis activation by epinephrine, glucagon or theophylline. The antilipolytic effect of pro-insulin was not blocked by Kunitz pancreatic trypsin inhibitor (KPTI). The-concentrations of proinsulin and insulin required to cause 50 per cent inhibition of epinephrine-stimulated lipolysis were estimated to be approximately 1 × 10−9 M and 3 × 10−11 M respectively. Proinsulin which had been reduced and allowed to reoxidize was found to have approximately the same antilipolytic activity as native proinsulin, indicating that the antilipolytic effect was due to proinsulin and not insulin contamination. The concentrations of proinsulin and insulin required to give 50 per cent of maximal stimulation of glucose oxidation were estimated to be approximately 5 × 10−9 M and 1 × 10−11 M respectively. The effect of proinsulin on glucose oxidation did not appear to be caused by any insulin contamination since reduced-reoxidized proinsulin had approximately the same activity as native proinsulin. KPTI had no effect on proinsulin-stimulated glucose oxidation in isolated fat cells, in contrast to the marked inhibitory effect observed using intact epididymal fat pad. This suggests that the KPTI-sensitive proteolytic activity of the epididymal fat pad is not located in the fat cell but in some other type cell or extracellular space.