The two tumor necrosis factor receptors mediate opposite effects on differentiation and glucose metabolism in human adipocytes in primary culture

Tumor necrosis factor-alpha (TNF) inhibits fat cell differentiation and may also mediate insulin resistance in adipocytes. Both TNF receptors are expressed in adipose tissue, but it is unknown how both receptors are involved in these biological functions. We therefore studied the effect of receptor-specific TNF muteins on adipose differentiation and insulin-stimulated glucose transport of in vitro differentiated human adipocytes in primary culture. Adipocyte precursor cells exposed to the 60-kDa TNF receptor (p60-TNFR)-specific TNF(R32W-S86T) showed a marked decrease in the percentage of differentiating cells in response to adipogenic factors as well as a reduction in peroxisome proliferator-activated receptor-gamma2 (PPARgamma2) messenger RNA (mRNA) and glycerophosphate dehydrogenase (GPDH) activity, but increased endogenous TNF mRNA expression. When cells were incubated with the p80-TNFR-specific TNF(D143N-A145R), adipogenesis and PPARgamma2 mRNA expression were stimulated, GPDH activity was unchanged, and TNF mRNA was completely suppressed. Insulin-stimulated 2-deoxy-D-glucose transport was inhibited by both muteins. The p60-TNFR-mediated inhibition increased continuously during 6 h of treatment and was associated with a down-regulation of glucose transporter-4 (GLUT4) mRNA and GLUT4 protein, whereas the p80-TNFR-specific mutein caused a transient increase in GLUT4 mRNA, but did not alter GLUT4 protein expression after a 24-h incubation. We conclude that p60-TNFR mediates the antiadipogenic effect as well as the down-regulation of GLUT4 by TNF, thereby leading to long-term inhibition of insulin-stimulated glucose transport. In contrast, activation of the p80-TNFR induces an adipogenic effect and transiently up-regulates GLUT4 expression. Here, the acute inhibition of insulin-stimulated glucose transport may be induced by interference with the insulin signaling pathway.     

Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects

PIKfyve is a phosphoinositide 5-kinase that can also act as a protein kinase. PIKfyve's role in acute insulin action has been suggested on the basis of its association with the insulin stimulatable phosphatidylinositol-3-kinase and the ability of acute insulin to recruit and phosphorylate PIKfyve on intracellular membranes of 3T3-L1 adipocytes. Here we have examined several classical insulin-regulated long- and short-term responses in insulin-sensitive cells expressing high levels of either active PIKfyve or kinase-dead mutants with a dominant-negative effect. Up-regulation of PIKfyve protein expression was documented in the early stages of differentiation of cultured 3T3-L1 fibroblasts into adipocytes and a kinase-dead mutant, PIKfyveDeltaK, introduced into the preadipocyte stage profoundly delayed the hormone-induced adipogenesis. Next, insulin-induced mitogenesis was markedly inhibited in HEK293 stable cell lines, inducibly expressing the dominant-negative kinase-dead PIKfyve(K1831E) mutant but not in cells expressing PIKfyve(WT). Similarly, expression of the dominant negative mutants PIKfyve(K1831E) or PIKfyveDeltaK strongly inhibited insulin-stimulated translocation of GLUT4 in 3T3-L1 adipocytes, or GLUT1-mediated glucose uptake in Chinese hamster ovary T cells expressing the human insulin receptor. Expression of PIKfyveDeltaK and PIKfyve(WT) in Chinese hamster ovary T cells decreased or increased, respectively, insulin-stimulated Akt phosphorylation at Ser473 but not at Thr308. Furthermore, a powerful inhibition of PIKfyve was documented at a very low concentration (ID(50) = 6 micro M) of the cell-permeable kinase inhibitor curcumin. When introduced into 3T3-L1 adipocytes, curcumin markedly inhibited insulin-induced GLUT4 translocation and glucose transport. Together these data indicate that PIKfyve enzymatic activity functions as a positive regulatory intermediate in insulin acute and long-term biological responses and identify Ser473 in Akt as one potential PIKfyve downstream target.     

Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb(1) has been shown to regulate peroxisome proliferator-activated receptor gamma activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb(1) on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb(1) significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 microM and a time of 3 h. In adipocytes, Rb(1) promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb(1) increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb(1)-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb(1) stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.     

Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes

Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.     

Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing

Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1-/- and Akt2-/- mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes. Loss of Akt1 alone slightly impaired insulin-mediated hexose transport activity but had no detectable effect on glycogen synthase kinase (GSK)-3 phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half of the insulin responsiveness. Combined depletions of Akt1 plus Akt2 in these cells even more markedly attenuated insulin action on glucose transporter 4 movements, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling, significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.     

Platelet-derived growth factor (PDGF) stimulates glucose transport in 3T3-L1 adipocytes overexpressing PDGF receptor by a pathway independent of insulin receptor substrates

Insulin is unique among growth factors and hormones in its ability to control metabolic functions such as the stimulation of glucose uptake and glucose transporter (GLUT4) translocation in physiological target tissues, such as muscle and adipose cells. Nonetheless, the mechanisms underlying this specificity have remained incompletely understood, particularly in view of the ability of some growth factors to mimic insulin-dependent early signaling events. In this study, we have probed the basis of insulin specificity by overexpressing in hormone-responsive 3T3-L1 adipocytes wild-type platelet-derived growth factor (PDGF) receptor (PDGFR)-beta and selected, informative mutant receptor proteins. We show that such adipocytes overexpressing wild-type PDGFR on exposure to cognate growth factor activate glucose transport, GLUT4 translocation, and the serine-threonine protein kinase Akt/protein kinase B to a degree comparable with that produced in response to insulin. In addition, PDGF elicits the robust generation of phosphatidylinositol-3,4,5-trisphosphate in vivo in PDGFR-overexpressing 3T3-L1 adipocytes. Expression of PDGFR-beta mutant proteins demonstrates that these responses require the presence of an intact phosphatidylinositol 3-kinase (PI3K)-binding site on the overexpressed PDGF receptor. Furthermore, PDGF stimulates these effects independent of insulin receptor substrate(IRS)-1 or IRS-2 tyrosine phosphorylation or docking to activated PI3K. These data demonstrate that 1) the basis of insulin-specific glucose transport in cultured adipocytes is the low level of receptors for other growth factors and 2) in the presence of adequate receptors, PDGF is fully capable of activating glucose transport in a manner requiring PI3K and subsequent phosphatidylinositol-3,4,5-trisphosphate accumulation but independent of insulin, insulin receptor, and IRS proteins.     

Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.     

Calpain system regulates the differentiation of adult primitive mesenchymal ST-13 adipocytes

The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-alpha (C/EBPalpha), peroxisome proliferator-activated receptor (PPAR)-gamma, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor alpha, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca(2+)-dependent degradation of PPARgamma and C/EBPalpha but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARgamma and C/EBPalpha. Under both conditions, calpain inhibitors almost completely prevented C/EBPalpha cleavage but partially blocked the decrease of PPARgamma. Two ubiquitous forms of calpain, mu- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of mu- but not m-calpain was found in the nucleus. Finally, stable dominant-negative mu-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARgamma and C/EBPalpha during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.     

Berberine stimulates glucose transport through a mechanism distinct from insulin

Berberine exerts a hypoglycemic effect, but the mechanism remains unknown. In the present study, the effect of berberine on glucose uptake was characterized in 3T3-L1 adipocytes. It was revealed that berberine stimulated glucose uptake in 3T3-L1 adipocytes in a dose- and time-dependent manner with the maximal effect at 12 hours. Glucose uptake was increased by berberine in 3T3-L1 preadipocytes as well. Berberine-stimulated glucose uptake was additive to that of insulin in 3T3-L1 adipocytes, even at the maximal effective concentrations of both components. Unlike insulin, the effect of berberine on glucose uptake was insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and SB203580, an inhibitor of p38 mitogen-activated protein kinase. Berberine activated extracellular signal-regulated kinase (ERK) 1/2, but PD98059, an ERK kinase inhibitor, only decreased berberine-stimulated glucose uptake by 32%. Berberine did not induce Ser473 phosphorylation of Akt nor enhance insulin-induced phosphorylation of Akt. Meanwhile, the expression and cellular localization of glucose transporter 4 (GLUT4) were not altered by berberine. Berberine did not increase GLUT1 gene expression. However, genistein, a tyrosine kinase inhibitor, completely blocked berberine-stimulated glucose uptake in 3T3-L1 adipocytes and preadipocytes, suggesting that berberine may induce glucose transport via increasing GLUT1 activity. In addition, berberine increased adenosine monophosphate-activated protein kinase and acetyl-coenzyme A carboxylase phosphorylation. These findings suggest that berberine increases glucose uptake through a mechanism distinct from insulin, and activated adenosine monophosphate-activated protein kinase seems to be involved in the metabolic effect of berberine.     

Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling

The 3 Akt protein kinase isoforms have critical and distinct functions in the regulation of metabolism, cell growth, and apoptosis, yet the mechanisms by which their signaling specificity is achieved remain largely unclear. Here, we investigated potential mechanisms underlying Akt isoform functional specificity by using Akt2-specific regulation of glucose transport in insulin-stimulated adipocytes as a model system. We found that insulin activates both Akt1 and Akt2 in adipocytes, but differentially regulates the subcellular distribution of these Akt isoforms. The greater accumulation of Akt2 at the plasma membrane (PM) of insulin-stimulated adipocytes correlates with Akt2-specific regulation of the trafficking of the GLUT4 glucose transporter. Consistent with this pattern, Akt constructs that do not accumulate at the PM to the same degree as Akt2 fail to regulate GLUT4 translocation to the PM, whereas enhancement of Akt1 PM association through mutation in Akt1 PH domain is sufficient to overcome Akt-isoform specificity in GLUT4 regulation. Indeed, we found that this distinct insulin-induced PM accumulation of Akt kinases is translated into a differential regulation by the Akt isoforms of AS160, a RabGAP that regulates GLUT4 trafficking. Our data show that Akt2 specifically regulates AS160 phosphorylation and membrane association providing molecular basis for Akt2 specificity in the modulation of GLUT4 trafficking. Together, our findings reveal the stimulus-induced subcellular compartmentalization of Akt kinases as a mechanism contributing to specify Akt isoform functions.     

MAP3K kinases and kidney injury

Mitogen-activated protein kinases (MAP kinases) are functionally connected kinases that regulate key cellular process involved in kidney disease such as all survival, death, differentiation and proliferation. The typical MAP kinase module is composed by a cascade of three kinases: a MAP kinase kinase kinase (MAP3K) that phosphorylates and activates a MAP kinase kinase (MAP2K) which phosphorylates a MAP kinase (MAPK). While the role of MAPKs such as ERK, p38 and JNK has been well characterized in experimental kidney injury, much less is known about the apical kinases in the cascade, the MAP3Ks. There are 24 characterized MAP3K (MAP3K1 to MAP3K21 plus RAF1, BRAF and ARAF). We now review current knowledge on the involvement of MAP3K in non-malignant kidney disease and the therapeutic tools available. There is in vivo interventional evidence clearly supporting a role for MAP3K5 (ASK1) and MAP3K14 (NIK) in the pathogenesis of experimental kidney disease. Indeed, the ASK1 inhibitor Selonsertib has undergone clinical trials for diabetic kidney disease. Additionally, although MAP3K7 (MEKK7, TAK1) is required for kidney development, acutely targeting MAP3K7 protected from acute and chronic kidney injury; and targeting MAP3K8 (TPL2/Cot) protected from acute kidney injury. By contrast MAP3K15 (ASK3) may protect from hypertension and BRAF inhibitors in clinical use may induced acute kidney injury and nephrotic syndrome. Given their role as upstream regulators of intracellular signaling, MAP3K are potential therapeutic targets in kidney injury, as demonstrated for some of them. However, the role of most MAP3K in kidney disease remains unexplored.     

A purine analog kinase inhibitor, calcium/calmodulin-dependent protein kinase II inhibitor 59, reveals a role for calcium/calmodulin-dependent protein kinase II in insulin-stimulated glucose transport

Olomoucine is known as a cyclin-dependent kinase inhibitor. We found that olomoucine blocked insulin's ability to stimulate glucose transport. It did so without affecting the activity of known insulin signaling proteins. To identify the olomoucine-sensitive kinase(s), we prepared analogs that could be immobilized to an affinity resin to isolate binding proteins. One of the generated analogs inhibited insulin-stimulated glucose uptake with increased sensitivity compared with olomoucine. The IC(50) for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 microM. To identify proteins binding to the analog, [(35)S]-labeled cell lysates prepared from 3T3-L1 adipocytes were incubated with analog chemically cross-linked to a resin support and binding proteins analyzed by SDS-PAGE. The major binding species was a doublet at 50-60 kDa, which was identified as calcium/calmodulin-dependent protein kinase II (CaMKII) by N-terminal peptide analysis and confirmed by matrix-assisted laser desorption ionization-mass spectrometry as the delta- and beta-like isoforms. To investigate CaMKII involvement in insulin-stimulated glucose uptake, 3T3-L1 adipocytes were infected with retrovirus encoding green fluorescent protein (GFP)-hemagluttinin tag (HA)-tagged CaMKII wild-type or the ATP binding mutant, K42M. GFP-HA-CaMKII K42M cells had less kinase activity than cells expressing wild-type GFP-HA-CaMKII. Insulin-stimulated glucose transport was significantly decreased (approximately 80%) in GFP-HA-CaMKII K42M cells, compared with nontransfected cells, and cells expressing either GFP-HA-CaMKII or GFP-HA. There was not a concomitant decrease in insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII K42M cells when compared with GFP-HA alone. However, insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII cells was significantly higher, compared with either GFP-HA or GFP-HA-CaMKII K42M cells. Our results implicate the involvement of CaMKII in glucose transport in a permissive role.     

叉头状转录因子O1对肥胖者内脏脂肪组织葡萄糖转运体4 mRNA及蛋白表达的影响

目的 利用RNAi技术沉默肥胖者内脏脂肪组织中叉头状转录因子O1(FOXO1)基因后检测葡萄糖转运体4(GLUT4)的mRNA和蛋白表达,探讨肥胖者FOXO1与胰岛素抵抗的关系及机制.方法 构建转录因子FOXO1特异性siRNA载体pSIREN-DNR-DsRed-siFOXO1(FOXO1-siRNA)质粒并鉴定;选取5例肥胖者大网膜脂肪组织利用脂质体为媒介转染FOXO1-siRNA质粒;半定量RT-PCR检测FOXO1 mRNA表达,测定抑制效率;半定量RT-PCR检测GLUT4 mRNA表达量,Westernblot检测GLUT4蛋白表达量.采用t检验进行统计分析.结果 成功构建FOXO1-siRNA质粒,转染此质粒的脂肪组织细胞FOXO1 mRNA表达水平与对照组相比下降约49%(P＜0.05),说明转染成功;FOXO1-siRNA质粒转染组GLUT4 mRNA较对照组增加约1.33倍(P=0.001),FOXO1-siRNA质粒转染组GLUT4蛋白较对照组增加约1.25倍(P＜0.05).结论 抑制肥胖者网膜内脏脂肪组织FOXO1基因的表达可引起GLUT4 mRNA及蛋白表达增加.     

Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes.

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.     

GLUT4 facilitates insulin stimulation and cAMP-mediated inhibition of glucose transport.

The glucose transporter isoform GLUT4 is found only in cells that exhibit insulin-sensitive glucose transport. To investigate the function of this transporter, L6 myoblasts were stably transfected with GLUT4 cDNA. GLUT4 underwent insulin-dependent movement to the cell surface in myoblasts overexpressing the transporter. One cell line (243-6) expressed sufficient levels of the GLUT4 protein to study insulin-dependent glucose transport. Unlike wild-type L6 cells, 243-6 myoblasts exhibited two features that are characteristic of differentiated muscle fibers and adipocytes in vivo: a large insulin-stimulated component of glucose transport and inhibition of this stimulated component by cAMP. Relative to normal L6 cells, 243-6 cells responded to insulin or insulin-like growth factor 1 with a 5-fold larger increase in 2-deoxy[3H]glucose uptake. N6,O2'-Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) did not inhibit transport in normal L6 myoblasts, which express only GLUT1, but inhibited IGF-1/insulin-stimulated transport by 50% in 243-6 cells. The effect of cAMP was investigated further by using Chinese hamster ovary cells transiently expressing GLUT1 and GLUT4. Bt2cAMP inhibited glucose transport only in Chinese hamster ovary cells expressing GLUT4. These results indicate that cAMP-mediated inhibition of glucose transport is dependent on expression of the GLUT4 isozyme.     

Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors

OBJECTIVE: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake. METHODS: Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1. RESULTS: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal. CONCLUSIONS: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.