Chronic and endogenous regulation of insulin receptors by catecholamines in adipocytes from patients with a phaeochromocytoma

Insulin binding in adipocytes from patients with a phaeochromocytoma (PH) approached that of the controls (C) at low and higher concentrations of unlabeled insulin. The apparent receptor affinity was unchanged (ED50: PH 0.50×10^–9M and C0.60×10^–9M). Scatchard analysis of the binding data using the negative cooperative model revealed a 46% decrease in the total number of receptors together with no changes in both K^–e (PH 0.55×10⁹M^–1 and C 0.36×10⁹M^–1) and K^–f (PH 0.13×10⁹ M^–1 and C 0.07×10⁹ M^–1). According to the two site model, an altered proportion in the two classes of insulin binding sites was detected. This was accompanied by a catecholamine-desensitization of the adipocytes to the antilipolytic action of insulin. These events could represent a final situation of a chronic and endogeneous regulation by high levels of catecholamines of insulin receptors in human adipose tissue.     

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.     

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.     

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.     

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.     

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.     

Expression of glucose transporters (GLUT 1 and GLUT 4) in primary cultured rat adipocytes: differential evolution with time and chronic insulin effect.

We previously reported that in cultured adipose cell lines insulin increased selectively the expression of Glut 1, in contrast to in vivo regulation where variations in insulinemia have been shown to affect only GLUT 4. We have addressed here the question of the long-term regulation of GLUT 1 and GLUT 4 in fat cells by using primary cultures of rat adipocytes. Epididymal fat cells were isolated by collagenase and cultured 4 days in DMEM supplemented with BSA 1%, FCS 1%, and glucose 10 mM. GLUT 1 and GLUT 4 proteins were assessed in total cellular membranes by Western blotting, using specific antibodies against their respective C-terminal peptides. GLUT 1 steadily increased over culture time to reach at day 3, a level 3-fold higher than the initial value. In contrast, GLUT 4 decreased sharply and stabilized at day 3, at 30% of the initial value. The changes in GLUT 1 and GLUT 4 mRNAs with culture time were parallel to changes in the corresponding proteins, suggesting a pre-translational level of regulation. The expression of the lipogenic enzyme, fatty acid synthetase (FAS), highly expressed in fat cell, decreased over time following a pattern closely parallel to that of GLUT 4. Chronic exposure to insulin added at day 2 had no effect on GLUT 4 expression but increased the expression of GLUT 1 and FAS by 70% and 36%, respectively. Glucose consumption was stable over 4 days of culture, while lactate production increased from 24 to 36% of glucose utilization, in agreement with the loss in FAS. Glucose consumption increased only slightly with insulin (+160%), in good keeping with the low levels of expression of both GLUT 4 and FAS in these cultured cells. These data indicate that culture alters oppositely the expression of GLUT 1 and GLUT 4 in rat adipocytes and suggest that factor(s) other than insulin predominate in their regulation in vivo.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

GLUT4在胰岛素调控葡萄糖转运中作用

机体的血糖平衡调节主要依赖于胰岛素,其中一个重要的机制是胰岛素通过调控GLUT4的囊泡运转来调节脂肪细胞和肌细胞对葡萄糖的摄取。由胰岛素受体介导的一系列磷酸化过程能调节一些关键的GLUT4转运相关蛋白质的活性,这些蛋白质包括小GTP酶、拴系复合体和囊泡融合体。而这些蛋白质又反过来通过内膜系统调节GLUT4储存囊泡的生成、滞留,并调控这些囊泡的靶向出胞方式。了解这些过程有助于解释2型糖尿病中胰岛素耐受的机制,并可能为糖尿病提供新的靶向治疗方法。     

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.     

PLCgamma participates in insulin stimulation of glucose uptake through activation of PKCzeta in brown adipocytes

Based on recent studies showing that PLCgamma associates to insulin receptor, we investigated its role in insulin stimulation of glucose transport in brown adipocytes. Insulin stimulation induced rapid PLCgamma association to phosphorylated insulin receptor, and activation of PLCgamma, as assessed by the mobilization of Ca(2+) from intracellular stores and by the production of the second messenger DAG. Both events are dependent on activation of PI3-kinase. Inhibition of PLCgamma activity either with the chemical compound U73122 or with an inhibitor peptide precluded insulin stimulation of glucose uptake, GLUT4 translocation, and actin reorganization, as wortmannin did. In contrast, the inactive analog U73343 did not have an inhibitory effect. Furthermore, translocation of GLUT4-GFP in response to insulin was completely abolished by cotransfection with a PLCgamma-inactive mutant in HeLa cells, a cell model sensitive to insulin that express PLCgamma. U73122 did not affect PI3-kinase nor Akt activation, but impaired PKCzeta activation by insulin, as wortmannin did. PLC activity renders two products, IP(3) and DAG, and DAG can be metabolized to PA by the action of DAG-kinase. Using the compound R54494, a DAG-kinase inhibitor, insulin-induced PKCzeta activation was also suppressed, this activity being restored by addition of PA. In summary, these data indicate that PLCgamma, activated at least partially by PI3-kinase, is a link between insulin receptor and PKCzeta through the production of PA and could mediate insulin-induced glucose uptake and GLUT4 translocation.     

Enhancement of insulin signaling pathway in adipocytes by oxovanadium(IV) complexes

Recently, we have found that some oxovanadium(IV) complexes are potent insulin-mimetic compounds for treating both type I and type II diabetic animals. However, the functional mechanism of oxovanadium(IV) complexes is not fully understood. In this report, we have shown that oxovanadium(IV)-picolinate complexes such as VO(pa)(2), VO(3mpa)(2), and VO(6mpa)(2) act on the insulin signaling pathway in 3T3-L1 adipocytes. Among them, VO(3mpa)(2) was found to be the highest potent activator in inducing not only the phosphotyrosine levels of both IRbeta and IRS but also the activation of downstream kinases in the insulin receptor, such as Akt and GSK3beta, which in turn translocated the insulin-dependent GLUT4 to the plasma membrane. Then, we examined whether or not oxovanadium(IV)-picolinates exhibit the hypoglycemic activity in STZ-induced diabetic mice, and found that VO(3mpa)(2) is more effective than the others in improving the hyperglycemia of the animals. Our present data indicate that both activation of insulin signaling pathway, which follows the GLUT4 translocation to the plasma membrane, and enhancement of glucose utilization by oxovanadium(IV) complexes cause the hypoglycemic effect in diabetic animals.     

Insulin‐Regulated Trafficking of GLUT4 Requires Ubiquitination

A major consequence of insulin binding its receptor on fat and muscle cells is translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the cell surface where it serves to clear glucose from the bloodstream. Sorting of GLUT4 into its insulin‐sensitive store requires the GGA [Golgi‐localized, γ‐ear‐containing, ADP ribosylation factor (ARF)‐binding] adaptor proteins, but the signal on GLUT4 to direct this sorting step is unknown. Here, we have identified a role for ubiquitination of GLUT4 in this process. We demonstrate that GLUT4 is ubiquitinated in 3T3‐L1 adipocytes, and that a ubiquitin‐resistant version fails to translocate to the cell surface of these cells in response to insulin. Our data support a model in which ubiquitination acts as a signal for the trafficking of GLUT4 from the endosomal/trans‐Golgi network (TGN) system into its intracellular storage compartment, from where it is mobilized to the cell surface in response to insulin.     

Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes

Chronic inflammation is closely associated with metabolic disorders such as obesity and type 2 diabetes, however, the underlying mechanism is unclear. Toll-like receptors (TLRs) play a key role in innate immune response as well as inflammatory signals. Here, we observed that mRNA level of TLR4 was induced during adipocyte differentiation and remarkably enhanced in fat tissues of obese db/db mice. In addition, activation of TLR4 with either LPS or free fatty acids stimulated NFkappaB signaling and expression of inflammatory cytokine genes, such as TNFalpha and IL-6 in 3T3-L1 adipocytes. Furthermore, we discovered that TLR4 activation in 3T3-L1 adipocytes provoked insulin resistance. Taken together, these results suggest that activation of TLR4 in adipocyte might be implicated in the onset of insulin resistance in obesity and type 2 diabetes.     

Insulin stimulates turnover of phosphatidylcholine in rat adipocytes

The present study investigated the effect of insulin on phosphatidylcholine turnover in rat adipocytes labelled to equilibrium with [¹⁴C]-choline. Insulin induced a rapid turnover of this major phospholipid that was maximal by 1 min and transient in nature. Following a 1 min stimulation of the cells with insulin at a maximally effective concentration (7 nM), a 4–6% decrease in the percentage of total cellular choline associated with this phospholipid was observed. This reflected a significant transient increase in the percentage of total cellular choline associated with phosphorylcholine, which together with diacylglycerol are the phospholipase C cleavage products of phosphatidylcholine. These effects were observed over a physiological range of insulin concentrations. No effect of insulin on any other choline phospholipid or metabolite (sphingomyelin, lysophophatidylcholine, glycerophosphocholine or choline) was seen. These results suggest that insulin stimulates a phospholipase C-mediated turnover of phosphatidylcholine in rat adipocytes. The rapid nature of this turnover suggests a potential role in signal transduction.     

Insulinomimetic Zn complex (Zn(opt)2) enhances insulin signaling pathway in 3T3-L1 adipocytes

Zinc (Zn) is an essential trace element with multiple regulatory functions, involving insulin synthesis, secretion, signaling and glucose transport. Since 2000, we have proposed that Zn complexes with different coordination environments exhibit high insulinomimetic and antidiabetic activities in type 2 diabetic animals. However, the molecular mechanism for the activities is still unsolved. The purpose of this study was to reveal the molecular mechanism of several types of Zn complexes in 3T3-L1 adipocytes, with respect to insulin signaling pathway. Obtained results shows that bis(1-oxy-2-pyridine-thiolato)Zn(II), Zn(opt)2, with S(2)O(2) coordination environment induced most strongly Akt/protein kinase B (Akt/PKB) phosphorylation, in which the optimal phosphorylation was achieved at a concentration of 25 microM, and this Zn(opt)2-induced Akt/PKB phosphorylation was inhibited by wortmannin at 100 nM. Further, the phosphorylation was maximal at 5-10 min stimulation, in agreement with the Zn uptake which was also maximal at 5-10 min stimulation. The Akt/PKB phosphorylation was in concentration- and time-dependent manners. Zn(opt)2 was also capable to translocate GLUT4 protein to the plasma membrane. We conclude that Zn(opt)2 was revealed to exhibit both insulinomimetic and antidiabetic activities by activating insulin signaling cascade through Akt/PKB phosphorylation, which in turn caused the GLUT4 translocation from the cytosol to the plasma membrane.     

Kaempferitrin inhibits GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes

Insulin stimulated GLUT4 (glucose transporter 4) translocation and glucose uptake in muscles and adipocytes is important for the maintenance of blood glucose homeostasis in our body. In this paper, we report the identification of kaempferitrin (kaempferol 3,7-dirhamnoside), a glycosylated flavonoid, as a compound that inhibits insulin stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. In the absence of insulin, we observed that addition of kaempferitrin did not affect GLUT4 translocation or glucose uptake. On the other hand, kaempferitrin acted as an inhibitor of insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes by inhibiting Akt activation. Molecular docking studies using a homology model of GLUT4 showed that kaempferitrin binds directly to GLUT4 at the glucose transportation channel, suggesting the possibility of a competition between kaempferitrin and glucose during the transport. Taken together, our data demonstrates that kaempferitrin inhibits GLUT4 mediated glucose uptake at least by two different mechanisms, one by interfering with the insulin signaling pathway and the other by a possible competition with glucose during the transport.     

游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。     

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.