Decreased insulin binding and antilipolytic response in adipocytes from patients with Cushing's syndrome

Human adipocytes from patients with chronic endogenous hypercortisolism (Cushing's syndrome) showed a statistically significant decrease in insulin binding at low unlabelled-insulin concentrations but no change in receptor numbers (Cushing's 180,000±48,000 (3) receptors/cell and controls 189,000±30,000 (7)) together with a fourfold decrease in apparent receptor affinity (ED50: Cushing's 2.25×10^–9 M and controls 0.57×10^–9 M) and a decreased sensitivity to the antilipolytic effect of insulin. These events could represent the final situation of a chronic and endogenous regulation by high levels of cortisol of insulin receptors in human adipose tissue.     

Biological effects of sulphated insulin in adipocytes and hepatocytes

Summary The binding affinity of sulphated insulin compared with unmodified, neutral insulin has been reported to be approximately four times lower in human and rat adipocytes but over twenty times lower in rat hepatocytes. In the present study the biological action of sulphated insulin was assesed in rat hepatocytes and human and rat adipocytes. To achieve half-maximal stimulation of fatty acid synthesis in rat hepatocytes about twenty one times higher concentrations of sulphated than neutral insulin were required (15.07±5.50 vs 0.71±0.34 nmol/l), this ratio being similar to the ratio of binding affinity in rat hepatocytes. In human adipocytes, half-maximal stimulation of initial rates of glucose uptake was observed at 11.6±5.1 vs 2.9±1.3 pmol/l for sulphated and neutral insulin respectively, and half-maximal inhibition of lipolysis at 31.0±13.5 vs 7.3+2.5 pmol/I respectively. These data are consistent with the four-fold lower binding affinity of sulphated insulin to human adipocytes. However, in rat adipocytes the biological potency of sulphated insulin was found to be much lower than anticipated from the binding data, half-maximal stimulation of initial rates of glucose uptake being observed at 757±299 vs 35±13 pmol/l respectively and half-maximal inhibition of lipolysis at 35.9±12.1 vs 1.5±0.5 pmol/l respectively. Thus, in rat adipocytes, approximately 22 times the concentration of sulphated insulin was required to achieve equivalent biological effect. A discrepancy between binding affinity and biological action with respect to sulphated insulin was identified in rat adipocytes but not human adipocytes nor rat hepatocytes suggesting differences in the binding-action linkage in these cells.     

Long lasting cadmium intake is associated with reduction of insulin receptors in rat adipocytes

The effects of chronic cadmium exposure on adipose tissue have not been extensively reported. In adult Wistar male rats we investigated in vivo effect of 6 weeks lasting cadmium intake in drinking tap water (CdCl₂ 9,7 mg/l). Insulin receptors in isolated adipocytes from epididymal fat and glucose transporter protein GLUT4 content in fat tissue plasma membranes were determined. Control and Cd treated rats had similar water intake with subsequent heavy augmentation of Cd content in liver of experimental animals. In comparison with controls, Cd intake did not influence body mass increment and fat cell size, but significantly increased serum glycemia and moderately elevated insulinemia. Cadmium intake significantly reduced (50%) both, total insulin receptors number and density of the receptors in fat cells. No differences in the content of GLUT4 in crude plasma membranes of adipose tissue were observed. Diminished insulin receptors in adipocytes could account for diabetogenic effect of long lasting cadmium intake.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Dexamethasone stimulates leptin release from human adipocytes: Unexpected inhibition by insulin

In the present study we have examined the effect of dexamethasone on ob gene mRNA expression and leptin release from isolated human subcutaneous adipocytes. Dexamethasone stimulated leptin release from cultured adipocytes in a time- and dose-dependent manner. A two-fold increase in leptin release was detectable by 36 h of treatment with 10⁻⁷ M dexamethasone. Leptin release was preceded by a significant 83±30% increase in ob mRNA after 24 h exposure to the compound. Co-incubation of cells with dexamethasone (10⁷ M) and insulin (10⁻⁷ or 10⁻⁹ M) completely blocked the dexamethasone-stimulated increase in ob mRNA and leptin release. These data demonstrate that insulin and glucocorticoids regulate leptin synthesis and release from human adipocytes in vitro. J. Cell. Biochem. 65:254–258. © 1997 Wiley-Liss, Inc.     

Persistent biologic actions of insulin on cultured fetal human fibroblasts

Summary Monolayer cultures of fetal human fibroblasts, preincubated in serum-free culture medium overnight (about 18 hr), were incubated with insulin (0.1 to 100 mU per ml), then washed and incubated in an insulin-free medium. The effect of insulin on glucose utilization, uridine incorporation into RNA and leucine incorporation into protein was maintained after removal of insulin and washing. For both glucose utilization and uridine incorporation into RNA, this effect was demonstrated at physiologic levels of insulin (0.1 mU per ml). When anti-insulin serum was added to the cultures after the cell preincubated with insulin were washed, this effect was greatly attenuated. This lasting effect of insulin was probably not due to nonspecifically bound insulin becoming available to the cells. Binding of¹²⁵I-monoiodoinsulin was examined in monolayer cultures of fetal human fibroblasts. When unlabeled insulin was present at about 1 mU per ml concentration, 50% displacement of monoiodoinsulin occured. When fibroblasts were incubated with monoiodoinsulin and then removed from the radioactive medium, initial dissociation of the bound hormone occurred rapidly but then reached a plateau. This prolonged insulin effect appears to result from persistent binding of insulin to its receptor. Supported in part by PHS Grants AM-02456, AM-05020 and AM-15312, by the Kroc Foundation and by the Diabetes Center (AM-17047). Supported in part by Research Career Development Award AM-47142 from NIAMDD.     

Dissociation of insulin binding from insulin stimulation of 2-deoxyglucose transport by ruthenium red

Ruthenium red increased specific insulin binding to isolated adipocytes 5.4 fold and 2.6 fold over binding determined in the absence and presence of Ca²⁺ and Mg²⁺. The increase in insulin binding was not accompanied by an increase in insulin sensitivity. The lack of effect of ruthenium red on insulin action argued strongly against an increase in intracellular Ca²⁺ as a potential messenger/transducer of insulin action and suggested that the enhancing effect of Ca²⁺ on insulin action was a result of increased receptor affinity.Abbreviations RR ruthenium red - BSA bovine serum albumin - Hepes 4-(2-hydroxyethyl-1-piperazineethane-sulphonic acid     

Demonstration of a neuropeptide Y-like immunoreactivity in human phaeochromocytoma extracts

Using a porcine neuropeptide Y directed radioimmunoassay it was shown that acid extracts of human phaeochromocytoma tumour tissue contained a neuropeptide Y-like peptide. Further fractionation and purification of this immunoreactivity showed that this human neuropeptide Y-like immunoreactivity was closely similar in molecular size and separation characteristics to porcine neuropeptide Y. The possible contribution of neuropeptide Y to the hypertension characterizing human phaeochromocytoma is discussed.     

Phenethyl isothiocyanate protects against H2O2-induced insulin resistance in 3T3-L1 adipocytes

Obesity is associated with systemic oxidative stress and leads to insulin resistance. Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has been shown to have beneficial effects in improving cellular defense activities against oxidative stress through activation of nuclear factor erythroid-2 related factor 2 (Nrf2) pathway. However, little evidence exists if the antioxidative activity has beneficial effects on glucose metabolism. Here, we tested the preventive potential of PEITC for impaired insulin-induced glucose uptake by oxidative stress in 3T3-L1 adipocytes. Treatment with PEITC increased the expression of antioxidative enzymes regulated by Nrf2 such as γ-glutamylcysteine-synthetase, heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1 and glutathione S-transferase, and reduced oxidative stress induced by H₂O₂. Furthermore, PEITC restored impaired insulin-stimulated glucose uptake, translocation of glucose transporter 4 and insulin signaling by H₂O₂. These results indicate that PEITC protected insulin-regulated glucose metabolism impaired by oxidative stress through the antioxidative activity in 3T3-L1 adipocytes.     

Quantitative analysis of secretome from adipocytes regulated by insulin

Adipocyte is not only a central player involved in storage and release of energy, but also in regulation of energy metabolism in other organs via secretion of peptides and proteins. During the pathogenesis of insulin resistance and type 2 diabetes, adipocytes are subjected to the increased levels of insulin, which may have a major impact on the secretion of adipokines. We have undertaken cleavable isotope-coded affinity tag （cICAT） and label-free quantitation approaches to identify and quantify secretory factors that are differentially secreted by 3T3-L1 adipocytes with or without insulin treatment. Combination of cICAT and label-free results, there are 317 proteins predicted or annotated as secretory proteins. Among these secretory proteins, 179 proteins and 53 proteins were significantly upregulated and down-regulated, respectively. A total of 77 reported adipokines were quantified in our study, such as adiponectin, cathepsin D, cystatin C, resistin, and transferrin. Western blot analysis of these adipokines confirmed the quantitative results from mass spectrometry, and revealed individualized secreting patterns of these proteins by increasing insulin dose. In addition, 240 proteins were newly identified and quantified as secreted proteins from 3T3-L1 adipocytes in our study, most of which were up-regulated upon insulin treatment. Further comprehensive bioinformatics analysis revealed that the secretory proteins in extracellular matrix-receptor interaction pathway and glycan structure degradation pathway were significantly upregulated by insulin stimulation.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine the in vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5-6 times more than the basal control. And the uptake of D-[3-3H]-glucose is linear as the logarithm of insulin concentration from 0.2 μg/L to 1.0 μg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, the in vitro biological activity of [B2-Lys]-insulin and [B3-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes

Incubation of intact rat adipocytes with physiological concentrations of catecholamines inhibits the specific binding of 125I-insulin and 125I-epidermal growth factor (EGF) by 40 to 70%. Affinity labeling of the alpha subunit of the insulin receptor demonstrates that the inhibition of hormone binding is directly reflective of a specific decrease in the degree of receptor occupancy. The stereospecificity and dose dependency of the binding inhibitions are typical of a classic beta 1-adrenergic receptor response with half-maximal inhibition occurring at 10 nM R-(-)-isoproterenol. Specific alpha-adrenergic receptor agonists and beta-adrenergic receptor antagonists have no effect, while beta-adrenergic receptor antagonists block the inhibition of 125I-insulin and 125I-EGF binding to receptors induced by beta-adrenergic receptor agonists. Further, these effects are mimicked by incubation of adipocytes with dibutyryl cyclic AMP or with 3-isobutyl-1-methylxanthine. The beta-adrenergic inhibition of both 125I-insulin and 125I-EGF binding is very rapid, requiring only 10 min of isoproterenol pretreatment at 37 degrees C for a maximal effect. Removal of isoproterenol by washing the cells in the presence of alprenolol leads to complete reversal of these effects. The inhibition of 125I-EGF binding is temperature dependent whereas the inhibition of 125I-insulin binding is relatively insensitive to the temperature of isoproterenol pretreatment. Scatchard analysis of 125I-insulin and 125I-EGF binding demonstrated that the decrease of insulin receptor-binding activity may be due to a decrease in the apparent number of insulin receptors while the inhibition of EGF receptor binding can be accounted for by a decrease in apparent EGF receptor affinity. The decrease in the insulin receptor-binding activity is physiologically expressed as a dose-dependent decrease of insulin responsiveness in the adipocyte with respect to two known responses, stimulation of insulin-like growth factor II receptor binding and activation of the glucose-transport system. These results demonstrate a beta-adrenergic receptor-mediated cyclic AMP-dependent mechanism for the regulation of insulin and EGF receptors in the rat adipocyte.     

Comparison of the insulin binding, uptake and endogenous insulin content in long- and short-term starvation in Tetrahymena

FITC-insulin binding and endogenous insulin content of Tetrahymena pyriformis, that had been 24 h or 30 min starved, continuously fed or re-fed after starvation was studied by flow cytometry and confocal microscopy. Long starvation elevated both insulin binding and endogenous insulin content of the cells. Short re-feeding after long starvation or short starvation after continuous feeding does not change the situation. Fixed cells also bind FITC-insulin, however, in this case long starvation reduces, and re-feeding after long starvation elevates, the binding, which means that hormone binding by receptors only differs from receptor binding and engulfment (in living cells). The increase of FITC-insulin content in living cells seems to be due to engulfment, rather than by receptor binding. The results point to the unicellular organism's requirement for insulin production and binding in a life-threatening stress situation.     

重组人胰岛素的纯化及其性质的初步研究

构建含有人胰岛素原基因的重组载体,并在大肠杆菌表达系统中进行高效表达。表达产物经变性、复性、凝胶过滤纯化后,再经胰蛋白酶和羧肽酶B酶切作用,产物经离子交换层析纯化得到重组人胰岛素,且具有天然生物学活性。     

Modulation of insulin action by vanadate: evidence of a role for phosphotyrosine phosphatase activity to alter cellular signaling

A number of vanadium compounds (vanadate, vanadyl sulfate, metavanadate) have insulin-mimicking actions bothin vitro andin vivo. They have multiple biological effects in cultured cells and interact directly with various enzymes. The inhibitory action on phosphoprotein tyrosine phosphatases (PTPs) and enhancement of cellular tyrosine phosphorylation appear to be the most relevant to explain the ability to mimic insulin. We demonstrated that in rat adipocytes both acute insulin effects, e.g. stimulation of IGF-II and transferrin binding and a chronic effect, insulin receptor downregulation, were stimulated by vanadate. Vanadate also enhanced insulin binding, particularly at very low insulin concentrations, associated with increased receptor affinity. This resulted in increased adipocyte insulin sensitivity. Finally vanadate augmented the extent of activation of the insulin receptor kinase by submaximal insulin concentrations. This was associated with a prolongation of the insulin biological response, lipogenesis, after removal of hormone.In conclusion: in rat adipocytes vanadate promotes insulin action by three mechanisms, 1) a direct insulin-mimetic action, 2) an enhancement of insulin sensitivity and 3) a prolongation of insulin biological response. These data suggest that PTP inhibitors have potential as useful therapeutic agents in insulin-resistant and relatively insulin-deficient forms of diabetes mellitus.     

Adenosine effects upon insulin action on lipolysis and glucose transport in human adipocytes

The dose response effect of a new adenosine analogue, GR 79236 (N-[1S trans-2-hydroxycyclopentyl] adenosine) upon insulin sensitivity was examined in human adipocytes. The influence of adenosine upon insulin sensitivity for suppression of lipolysis and stimulation of glucose transport was examined. Removal of adenosine by use of adenosine deaminase stimulated lipolysis to the same extent as did 10^–9 M noradrenaline. GR79236 brought about dose dependent inhibition of lipolysis with half-maximal effect at 11.3±7.8×10^–9 M. When lipolysis was stimulated by noradrenaline alone the subsequent inhibition of lipolysis brought about by GR79236 was significantly greater than that of insulin. To examine adenosine effects on the insulin signalling pathway separately from those on lipolysis, the insulin sensitivity of glucose transport was examined. Removal of adenosine brought about a small but significant increase in the concentration of insulin required for half-maximal stimulation of glucose transport. Adenosine agonists offer promise as new agents for the modulation of metabolism in diabetes and other states of insulin resistance.     

Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors

Madin-Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites (Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen, nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface-labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipitates were analyzed on SDS-gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells wee incubated first with serum containing antibodies to the insulin receptor and then with 125I-protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin-like activity were tested on MDCK cells. The insulinomimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [3H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin-insensitive. However, since insulin-associated responses could be elicited by some insulin mimickers, the post-receptor limb of response in MDCK cells was apparently intact.