Sex steroid hormones induce acylation stimulating protein resistance in 3T3-L1 adipocytes

Acylation stimulating protein (ASP) stimulates triglyceride synthesis and glucose transport via its receptor C5L2. In human studies, ASP is increased in insulin resistant states such as obesity, diabetes, polycystic ovary syndrome and late pregnancy (the latter two associated with altered sex hormones). The aims were (i) to evaluate ASP response and C5L2 expression following treatment with sex steroid hormones and (ii) to identify mechanisms of ASP resistance using 3T3-L1 adipocytes and preadipocytes. Overnight incubation with physiological progesterone (PROG) concentrations induced dose-dependent inhibition of ASP-stimulated glucose transport in adipocytes (188 +/- 11% +ASP, 100 +/- 4% control, 129 +/- 18% to 85 +/- 7% [ASP + PROG 10(-8) to 10(-6) M] and preadipocytes (263 +/- 18% +ASP, 100 +/- 3% control, 170 +/- 11% to 167 +/- 4% [ASP + PROG 10(-8) to 10(-6) M]), while estradiol and testosterone (TEST) were effective only at the highest concentration (10(-6) M). In adipocytes, dose-dependent maximal C5L2 mRNA decreases were 39-75% (P = 0.003), with decreased cell-surface C5L2 of -22% and -27% (10(-6) M PROG and TEST, respectively) with no change in preadipocytes. Adipocytes treated with PROG displayed decreases in G proteins: Gbeta (-55%), Galphaq/11 (-56%) as well as complete inhibition of ASP stimulation. PROG significantly decreased basal levels of phosphorylated PKCalpha (p-PKCalpha) while there was no change in p- PKCzeta. ASP increased p-PKCalpha and PKCzeta to 161% (P < 0.0.001) and 160% (P < 0.01), a stimulation effectively blocked by PROG (10(-8) and 10(-6) M) and TEST (10(-6) M). Sex steroid hormone-induced ASP resistance via C5L2 may contribute to altered adipose tissue function and insulin resistance phenotype in humans.     

C5L2 is a functional receptor for acylation-stimulating protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.     

Targeting the signaling pathway of acylation stimulating protein

Acylation stimulating protein (ASP; C3adesArg) stimulates triglyceride synthesis (TGS) and glucose transport in preadipocytes/adipocytes through C5L2, a G-protein-coupled receptor. Here, ASP signaling is compared with insulin in 3T3-L1 cells. ASP stimulation is not Galpha(s) or Galpha(i) mediated (pertussis and cholera toxin insensitive), suggesting G(alphaq) as a candidate. Phospholipase C (PLC) is required, because the Ca(2+) chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and the PLC inhibitor U73122 decreased ASP stimulation of TGS by 93.1% (P < 0.0.001) and 86.1% (P < 0.004), respectively. Wortmannin and LY294002 blocked ASP effect by 69% (P < 0.001) and 116.1% (P < 0.003), respectively, supporting phosphatidylinositol 3-kinase (PI3K) involvement. ASP induced rapid, transient Akt phosphorylation (maximal, 5 min; basal, 45 min), which was blocked by Akt inhibition, resembling treatment by insulin. Downstream of PI3K, mamalian target of rapaycin (mTOR) is required for insulin but not ASP action. By contrast, both ASP and insulin activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK(1/2)) pathway, with rapid, pronounced increases in ERK(1/2) phosphorylation, effects partially blocked by PD98059 (64.7% and 65.9% inhibition, respectively; P < 0.001). Time-dependent (maximal, 30 min) transient calcium-dependent phospholipase A(2) (cPLA(2))(-Ser505) phosphorylation (by MAPK/ERK(1/2)) was demonstrated by Western blot analysis. ASP signaling involves sequential activation of PI3K and PLC, with downstream activation of protein kinase C, Akt, MAPK/ERK(1/2), and cPLA(2), all of which leads to an effective and prolonged stimulation of TGS.     

The ASP receptor C5L2 is regulated by metabolic hormones associated with insulin resistance.

Acylation-stimulating protein (ASP) and interaction with its receptor C5L2 influences adipocyte metabolism. We examined insulin resistance and differentiation-mediated regulation of C5L2 and the mechanistic impact on both C5L2 cell-surface protein and ligand binding to the receptor. C5L2 mRNA increased 8.7-fold with differentiation in 3T3-L1 cells (p < 0.0001) by day 9. In preadipocytes, insulin and dexamethasone increased C5L2 mRNA (1 micromol/L insulin resulted in a 2.6-fold increase, p < 0.01; 10 nmol/L dexamethasone resulted in a 17.9-fold increase, p < 0.01) and C5L2 cell-surface protein (100 nmol insulin resulted in a 2.7-fold increase, p < 0.001; 10 nmol/L dexamethasone resulted in a 2.8-fold increase, p < 0.001). In adipocytes, 100 nmol/L insulin increased C5L2 mRNA and ASP binding (respectively, 1.3-fold, p < 0.01; and 2.4-fold, p < 0.05). Dexamethasone decreased ligand binding (-60%, p < 0.02) without changing mRNA. Tumor necrosis factor alpha decreased C5L2 mRNA (-88% in preadipocytes and -38% in adipocytes, p < 0.001), C5L2 cell-surface protein (-53% in preadipocytes, p < 0.0001), and ASP binding (-60% and -49% in, respectively, preadipocytes and adipocytes, p < 0.05). Conversely, 1 micromol/L and 10 nmol/L rosiglitazone increased, respectively, C5L2 mRNA (9.3-fold, p < 0.0001) and ASP binding (2.4-fold, p < 0.05). Thus, C5L2 mRNA increases with differentiation, insulin, and thiazolidinedione treatment, and decreases with tumor necrosis factor alpha, all of which results in functional changes in ASP-C5L2 response and may have implications for human metabolism.     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression

Evidence has accumulated that ginseng and its main active constituents, ginsenosides, possess anti-diabetic and insulin-sensitizing properties which may be partly realized by regulating adipocyte development and functions. In the present study, we explored the effect of ginsenoside Rb(1), the most abundant ginsenoside in ginseng root, on adipogenesis of 3T3-L1 cells. We found that with standard differentiation inducers, ginsenoside Rb(1) facilitated adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner; 10 microM Rb(1) increased lipid accumulation by about 56%. Treatment of differentiating adipocytes with 10 microM Rb(1) increased the expression of mRNA and protein of PPARgamma(2) and C/EBPalpha, as well as mRNA of ap2, one of their target genes. After the treatment of differentiating adipocytes with Rb(1), basal and insulin-mediated glucose uptake was significantly augmented, accompanied by the up-regulation of mRNA and protein level of GLUT4, but not of GLUT1. In addition, ginsenoside Rb(1) also inhibited the proliferation of preconfluent 3T3-L1 preadipocytes. Our data indicate that anti-diabetic and insulin-sensitizing activities of ginsenosides, at least in part, are involved in the enhancing effect on PPARgamma2 and C/EBPalpha expression, hence promoting adipogenesis.     

3T3-L1 adipocyte glucose transporter (HepG2 class): sequence and regulation of protein and mRNA expression by insulin, differentiation, and glucose starvation

A glucose transporter cDNA (GLUT) clone was isolated from mouse 3T3-L1 adipocytes and sequenced. The nucleotide and deduced amino acid sequences were, respectively, 95 and 99% homologous to those of the rat brain transporter. The mouse cDNA and a polyclonal antibody recognizing the corresponding in vitro translation product were used to compare changes in transporter mRNA and protein levels during differentiation, glucose starvation, and chronic insulin exposure of 3T3-L1 preadipocytes. The respective cellular content of transporter mRNA and protein were increased 6.6- and 7.8-fold during differentiation, and 3.8- and 2.5-fold from chronic insulin exposure of differentiated adipocytes. Glucose starvation increased transporter mRNA and protein levels 2.2- and 3.5-fold in undifferentiated preadipocytes and 1.8- and 3.1-fold in differentiated adipocytes. Starvation of undifferentiated cells completely converted the native transporter to an incompletely glycosylated form, while increasing basal transport rates 4.5-fold. Either full glycosylation is not required to produce a functionally active transporter, or starvation causes a unique predifferentiation induction of the normally absent "responsive" transporter. The changes in transporter protein expression elicited by differentiation were attributed primarily to increased rates of transporter synthesis, while the disproportionate changes in mRNA and protein expression from chronic insulin treatment and starvation suggested these conditions increase synthesis and decrease turnover rates in regulating transporter protein expression. Although chronic insulin exposure and glucose starvation each raised the expression of transporter protein greater than 3-fold and basal transport rates 2.5- to 4.5-fold, no significant increase in the insulin responsiveness of 3T3-L1 preadipocytes or differentiated adipocytes was observed. Thus, the changes in the transporter mRNA and protein expression observed in this study were most consistent with their being associated with the regulated expression of a basal or low level insulin-responsive transporter.     

Nigericin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes

We used nigericin, a K+/H+ exchanger, to test whether glucose transport in 3T3-L1 adipocytes was modulated by changes in intracellular pH. Our results showed that nigericin increased basal but decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner. Whereas the basal translocation of GLUT1 was enhanced, insulin-stimulated GLUT4 translocation was inhibited by nigericin. On the other hand, the total amount of neither transporter protein was altered. The finding that insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase) activity was not affected by nigericin implies that nigericin exerted its inhibition at a step downstream of PI 3-kinase activation. At maximal dose, nigericin rapidly lowered cytosolic pH to 6.7; however, this effect was transient and cytosolic pH was back to normal in 20 min. Removal of nigericin from the incubation medium after 20 min abolished its enhancing effect on basal but had little influence on its inhibition of insulin-stimulated glucose transport. Moreover, lowering cytosolic pH to 6.7 with an exogenously added HCl solution had no effect on glucose transport. Taken together, it appears that nigericin may inhibit insulin-stimulated glucose transport mainly by interfering with GLUT4 translocation, probably by a mechanism not related to changes in cytosolic pH.     

Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.     

Tannins present in Cichorium intybus enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adipocytes through PTP1B inhibition

Insulin resistance is a fundamental aspect for the etiology of non-insulin dependent diabetes mellitus (NIDDM) and has links with a wide array of secondary disorders including weight gain and obesity. The present study analyzes the effect of Cichorium intybus methanolic (CME) extract on glucose transport and adipocyte differentiation in 3T3-L1 cells by studying the radiolabelled glucose uptake and lipid accumulation assays, respectively. By performing detannification (CME/DT), the role of tannins present in CME on both the activities was evaluated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a dose-dependent response. Glucose uptake profile in the presence of PI3K and IRTK inhibitors (Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptake in inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME. Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression analysis revealed the unique behavior of CME and CME/DT. The duality exhibited by C. intybus through adipogenesis inhibition and PPARgamma up regulation is of interest. Current observation concludes that the activities possessed by C. intybus are highly desirable for the treatment of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1 adipocytes.     

Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes

In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 μg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.     

促酰化蛋白(ASP)诱导3T3-L1前脂肪细胞分化

促酰化蛋白 (ASP)代替经典激素“鸡尾酒”诱导法中胰岛素 ,通过形态学观察、油红染色分化百分比测定、脂肪细胞甘油三酯合成率和甘油三酯总量测定 ,并与经典激素“鸡尾酒”法诱导前脂肪细胞分化情况比较 ,探讨ASP是否具有诱导 3T3 L1前脂肪细胞分化作用 .ASP组诱导分化第 6d ,3T3 L1前脂肪细胞变大、变圆 ,出现大量脂肪滴 ,形态由前脂肪细胞向成熟脂肪细胞转变 ;随着诱导分化时间延长 ,胞浆中脂滴进一步积累 .分化 9d时 ,3T3 L1前脂肪细胞分化完全 .油红染色结果显示 ,ASP组分化率很高 (85 % ) ,与胰岛素组分化率 (90 % )相似 ,明显高于IBMX +DEX组 (4 0 % ) .ASP不仅促进 3T3 L1前脂肪细胞形态向成熟脂肪细胞转化 ,同时促进细胞中甘油三酯的合成和积累 .ASP组诱导分化第 3d时 ,脂肪细胞甘油三酯合成率明显高于对照组和IBMX +DEX组 ,但仍低于胰岛素组 ;在分化第 6d和第 9d时 ,ASP组甘油三酯合成率进一步升高 ,与对照组和IBMX +DEX组相比差异有极显著性 ,与胰岛素组相比无显著性差异 .ASP组诱导分化 3d时 ,脂肪细胞中甘油三酯总量明显高于对照组和IBMX +DEX组 ;分化 6d和 9d时 ,甘油三酯总量进一步升高 ,与对照组和IBMX +DEX组相比差异有极显著性 ,而与胰岛素组相比无显著性差异 .结果表明 ,新型脂源性激     

The short-circuit current of the ileum, but not the colon, is altered in the streptozotocin diabetic rat

Ion transport in control and streptozotocin-diabetic rat colon and ileum was studied using the Ussing chamber technique. No differences were observed between control and diabetic colonic mucosal short-circuit current under either basal or carbachol (100 nmol/L-1 micromol/L)-stimulated or prostaglandin E2 (100 nmol/L-1 micromol/L)-stimulated conditions. Similarly to colonic tissues, no differences in the short circuit current in either carbachol-stimulated or prostaglandin E2-stimulated tissues were observed between control and diabetic ileal mucosa. The basal diabetic ileal short circuit current (99.58 +/- 22.67 microA) was significantly greater than that of control ileal tissues (29.67 +/- 4.45 microA). This difference was abolished by the sodium-glucose-cotransporter inhibitor, phloridzin (50 micromol/L) (118.00 +/- 28.09 microA vs. 25.60 +/- 4.59 microA) and was also prevented by the replacement of glucose with mannitol in the buffer bathing the apical side of the tissue (control: 17.05 +/- 5.85 microA vs. 17.90 +/- 3.10 microA). Acetazolamide (450 micromol/L; a carbonic anhydrase inhibitor), amiloride, and bumetanide (100 micromol/L each; Na+-channel blockers), piroxicam (50 micromol/L; a COX1 cyclooxygenase inhibitor), and ouabain (1 mmol/L; a K+ transport inhibitor) had no effect on the basal short circuit current of either control or diabetic ileal tissues. This indicated that the alteration in the basal short circuit current of diabetic ileal tissues was due to a change in cellular glucose transport, whereas the evoked changes in short circuit current were unaffected by the diabetic state.     

Site-specific differences of insulin action in adipose tissue derived from normal prepubertal children

Body fat distribution determines obesity-related morbidity in adults but little is known of the aetiology or pathophysiology in children. This study investigates differences in insulin-mediated metabolism in primary cell cultures of subcutaneous and visceral preadipocytes derived from prepubertal children. The impact of differentiation and responses to TNFalpha exposure was also investigated. Proliferation rates were greater in subcutaneous versus visceral preadipocytes (41 h3 versus 69 h4; P=0.008). Insulin caused a dose-dependent increase in GSK-3 phosphorylation and an increase in MAPK phosphorylation over time, with increased sensitivity in subcutaneous preadipocytes. Post-differentiation, dose-dependent increases in GSK-3 phosphorylation were maintained, while MAPK phosphorylation was identical in both subtypes. No changes were observed in insulin receptor abundance pre-/post-differentiation. GLUT4 abundance was significantly increased in visceral versus subcutaneous adipocytes by 76(4)%; P=0.03), coincidental with increased insulin-stimulated 2-deoxy-glucose transport (+150(26)% versus +79(10)%; P=0.014) and further elevated by acute exposure to TNFalpha (+230(52)%; P=0.019 versus +123(24)%; P=0.025, respectively). TNFalpha also significantly increased basal glucose transport rates (+44(14)%; P=0.006 versus +34(11)%; P=0.007) and GLUT1 localisation to the plasma membrane. These data establish site-specific differences in subcutaneous and visceral fat cells from children. Responses to insulin varied with differentiation and TNFalpha exposure in the two depots, consistent with parallel changes in GLUT1/4 abundance and localisation.     

Phorbol esters imitate in rat fat-cells the full effect of insulin on glucose-carrier translocation, but not on 3-O-methylglucose-transport activity.

Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucose-transport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 mu units/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7 +/- 1.3%, insulin 60 +/- 3.1%, TPA 22 +/- 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 +/- 1.0, insulin 13.4 +/- 2.0, TPA 12.7 +/- 2.7; low-density membranes, basal 12.8 +/- 2.1, insulin 6.3 +/- 0.9, TPA 8.9 +/- 0.7; high-density membranes, 6.9 +/- 1.1; insulin 12.5 +/- 1.0, TPA 8.1 +/- 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.     

Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.     

Discoidin domain receptor 2 impairs insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation and glucose uptake in 3T3-L1 adipocytes.

Differentiation of preadipocytes into functional adipocytes depends on early proliferative events (mitotic clonal expansion) and extracellular matrix interactions. We report that discoidin domain receptor (DDR) 2, a novel adhesion receptor, is expressed in 3T3-L1 preadipocytes and is downregulated during the early phase of adipogenesis. DDR2 overexpression (DDR2-L1 preadipocytes) reduced subconfluent proliferation by 56% (p<0.001) and insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 by 34% (p<0.05). The mitotic clonal expansion phase of differentiating confluent DDR2-L1 preadipocytes was impaired by approximately 25% (p<0.05). Although induction of peroxisome proliferator-activated receptor gamma, fatty acid synthase, and adiponectin was not altered, the resulting adipocytes were 55% larger (p<0.05), and contained 66% more triacylglycerol (p<0.01). The induction of CCAAT/enhancer binding protein alpha was reduced by 37% (p<0.05), correlating with a similar reduction in insulin-stimulated IRS-1 tyrosine phosphorylation and glucose transport in DDR2-L1 adipocytes (decreases of 22% and 27%, respectively; p<0.05 for both). Our data show that DDR2 is expressed in adipose cells and that its overexpression leads to insulin resistance.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo

Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.     

Mannose, glucosamine and inositol monophosphate inhibit the effects of insulin on lipogenesis. Further evidence for a role for inositol phosphate-oligosaccharides in insulin action.

The mechanism of insulin signalling is not yet understood in detail. Recently, a role for inositol phosphate (IP)-oligosaccharides as second messengers transmitting the insulin signal at the post-kinase level was proposed. To evaluate this hypothesis further, we studied whether IP-oligosaccharides isolated from 'haemodialysate' have insulin-like activity. We found that these compounds mimic, in a dose-dependent fashion, the following effects of insulin in adipocytes. (1) Lipogenesis. Incorporation of [3H]glucose into lipids (expressed in nmol/min per 10(6) cells): basal, 0.74 +/- 0.05; insulin (1 mu unit/ml), 4.43 +/- 0.21; IP-oligosaccharide (2 micrograms/ml), 4.07 +/- 0.19. (2) Inhibition of isoprenaline (isoproterenol) (1 microM)-stimulated cyclic AMP levels and lipolysis. Cyclic AMP (pmol/10(5) cells): basal 0.84 +/- 0.05; isoprenaline, 4.03 +/- 0.19; isoprenaline + insulin (200 mu units/ml), 2.06 +/- 0.7; isoprenaline + IP-oligosaccharides (2 micrograms/ml), 2.4 +/- 0.29. Inhibition of lipolysis (mumol of glycerol/mg of protein): isoprenaline (1 microM), 166 +/- 11; isoprenaline+insulin (150 mu units/ml), 53 +/- 3.5; isoprenaline+IP-oligosaccharides (2 micrograms/ml), 58 +/- 5. (3) Stimulation of 3-O-methylglucose transport; basal, 9 +/- 3%; insulin (1 mu unit/ml), 67 +/- 4%, IP-oligosaccharides (2 micrograms/ml), 54 +/- 2%. To identify the active molecules of the IP-oligosaccharide fraction, competition experiments were performed. IP-oligosaccharide effects on lipogenesis were blocked by inositol monophosphate, glucosamine and mannose. In contrast, these compounds did not inhibit IP-oligosaccharide effects on membrane-mediated functions (3-O-methylglucose transport, cyclic AMP levels, lipolysis). We also found that the effect of insulin on lipogenesis was blocked by mannose, glucosamine and inositol monophosphate, whereas the insulin effects on 3-O-methylglucose, cyclic AMP and lipolysis were unaffected. The following conclusions were reached. (1) IP-oligosaccharides mimic the major metabolic effects of insulin in adipocytes. This is consistent with the proposed role of IP-oligosaccharides as second messengers of certain insulin effects. (2) Mannose and glucosamine are functionally important sugar residues for the effect of IP-oligosaccharide on lipogenesis. (3) The observation that mannose, inositol monophosphate and glucosamine block the action of insulin of on lipogenesis supports a role of mannose- and glucosamine-containing IP-oligosaccharides as second messengers for this insulin effect.