Clinical outcomes with pemetrexed-based systemic therapies in RET-rearranged lung cancers.

BACKGROUND: RET rearrangements are targetable, oncogenic lung cancer drivers. While previous series have shown durable clinical benefit with pemetrexed-based therapies in ALK- and ROS1-rearranged lung cancers, the benefits of pemetrexed-based treatments in patients with RET-rearranged lung cancers relative to other genomic subsets have not previously been explored. PATIENTS AND METHODS: A retrospective review of patients with pathologically confirmed stage IIIB/IV lung adenocarcinomas and evidence of a RET, ROS1, or ALK rearrangement, or a KRAS mutation was conducted. Patients were eligible if they received treatment with pemetrexed alone or in combination. The primary outcome of progression-free survival (PFS), and secondary outcomes of overall response rate (ORR, RECIST v1.1), time to progression (TTP), and time to treatment discontinuation were compared between RET-rearranged and groups of ROS1-rearranged, ALK-rearranged, and KRAS-mutant lung cancers. RESULTS: We evaluated 104 patients. Patients with RET-rearranged lung cancers (n = 18) had a median PFS of 19 months [95% confidence interval (CI) 12-not reached (NR)] that was comparable with patients with ROS1- (23 months, 95% CI 14-NR, n = 10) and ALK-rearranged (19 months, 95% CI 15-36, n = 36) lung cancers, and significantly improved compared with patients with KRAS-mutant lung cancers (6 months, 95% CI 5-9, P < 0.001, n = 40). ORR (45%), median TTP (20 months, 95% CI 17-NR), and median time to treatment discontinuation (21 months, 95% CI 6-NR) in patients with RET-rearranged lung cancers were not significantly different compared with patients with ALK- and ROS1-rearranged lung cancers, and improved compared with patients with KRAS-mutant lung cancers. CONCLUSION: Durable benefits with pemetrexed-based therapies in RET-rearranged lung cancers are comparable with ALK- and ROS1-rearranged lung cancers. When selecting therapies for patients with RET-rearranged lung cancers, pemetrexed-containing regimens should be considered.

An integrated genomic analysis of lung cancer reveals loss of DUSP4 in EGFR-mutant tumors.

To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.

Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells.

AIMS/HYPOTHESIS: The aim of this study was to determine whether adiponectin elicits glucose uptake via increased GLUT4 translocation and to investigate the metabolic fate of glucose in skeletal muscle cells treated with globular adiponectin. MATERIALS AND METHODS: Basal and insulin-stimulated 2-deoxy-D: -[(3)H]glucose uptake, cell surface myc-tagged GLUT4 content, production of (14)CO(2) by oxidation of D: -[U-(14)C]glucose and [1-(14)C]oleate, and incorporation of D: -[U-(14)C]glucose into glycogen and lactate were measured in the presence and absence of globular adiponectin. RESULTS: RT-PCR and Western blot analysis revealed that L6 cells and rat skeletal muscle cells express AdipoR1 mRNA and protein. Globular adiponectin increased both GLUT4 translocation and glucose uptake by increasing the transport V(max) of glucose without altering the K(m). Interestingly, the incorporation of D: -[U-(14)C]glucose into glycogen under basal and insulin-stimulated conditions was significantly decreased by globular adiponectin, whereas lactate production was increased. Furthermore, globular adiponectin did not affect glucose oxidation, but enhanced phosphorylation of AMP kinase and acetyl-CoA carboxylase, and fatty acid oxidation. CONCLUSIONS/INTERPRETATION: The present study is the first to show that globular adiponectin increases glucose uptake in skeletal muscle cells via GLUT4 translocation and subsequently reduces the rate of glycogen synthesis and shifts glucose metabolism toward lactate production. These effects are consistent with the increased phosphorylation of AMP kinase and acetyl-CoA carboxylase and oxidation of fatty acids induced by globular adiponectin.

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase.

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t(1/2)=2.5 min) preceded the stimulation of 2-deoxyglucose uptake (t(1/2)=6 min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22 degrees C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40-60%. Pretreatment with SB203580 lowered the apparent transport V(max) of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent K(m) for either hexose. The IC(50) values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2 microM respectively, and correlated with the IC(50) for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4 microM, respectively). Insulin caused a dose- (EC(50)=15 nM) and time- (t(1/2)=3 min) dependent increase in p38 MAPK phosphorylation, which peaked at 10 min (2.3+/-0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38 alpha (2.6+/-0.3-fold) and p38 beta (2.3+/-0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport.

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.

High leptin levels acutely inhibit insulin-stimulated glucose uptake without affecting glucose transporter 4 translocation in l6 rat skeletal muscle cells.

Obesity is a major risk factor for the development of insulin resistance, characterized by impaired stimulation of glucose disposal into muscle. The mechanisms underlying insulin resistance are unknown. Here we examine the direct effect of leptin, the product of the obesity gene, on insulin-stimulated glucose uptake in cultured rat skeletal muscle cells. Preincubation of L6 myotubes with leptin (2 or 100 nM, 30 min) had no effect on basal glucose uptake but reduced insulin-stimulated glucose uptake. However, leptin had no effect on the insulin-induced gain in myc-tagged glucose transporter 4 (GLUT4) appearance at the cell surface of L6 myotubes. Preincubation of cells with leptin also had no effect on insulin-stimulated tyrosine phosphorylation of insulin receptor, IRS-1 and IRS-2, phosphatidylinositol 3-kinase activity, or Akt phosphorylation. We have previously shown that insulin regulates glucose uptake via a signaling pathway sensitive to inhibitors of p38 MAP kinase. Here, leptin pretreatment reduced the extent of insulin-stimulated p38 MAP kinase phosphorylation and phosphorylation of cAMP response element binder, a downstream effector of p38 MAP kinase. These results show that high leptin levels can directly reduce insulin-stimulated glucose uptake in L6 muscle cells despite normal GLUT4 translocation. The mechanism of this effect could involve inhibition of insulin-stimulated p38 MAP kinase and GLUT4 activation.

The antihyperglycemic drug alpha-lipoic acid stimulates glucose uptake via both GLUT4 translocation and GLUT4 activation: potential role of p38 mitogen-activated protein kinase in GLUT4 activation.

The cofactor of mitochondrial dehydrogenase complexes and potent antioxidant alpha-lipoic acid has been shown to lower blood glucose in diabetic animals. alpha-Lipoic acid enhances glucose uptake and GLUT1 and GLUT4 translocation in 3T3-L1 adipocytes and L6 myotubes, mimicking insulin action. In both cell types, insulin-stimulated glucose uptake is reduced by inhibitors of p38 mitogen-activated protein kinase (MAPK). Here we explore the effect of alpha-lipoic acid on p38 MAPK, phosphatidylinositol (PI) 3-kinase, and Akt1 in L6 myotubes. alpha-Lipoic acid (2.5 mmol/l) increased PI 3-kinase activity (31-fold) and Akt1 (4.9-fold). Both activities were inhibited by 100 nmol/l wortmannin. alpha-Lipoic acid also stimulated p38 MAPK phosphorylation by twofold within 10 min. The phosphorylation persisted for at least 30 min. Like insulin, alpha-lipoic acid increased the kinase activity of the alpha (2.8-fold) and beta (2.1-fold) isoforms of p38 MAPK, measured by an in vitro kinase assay. Treating cells with 10 micromol/l of the p38 MAPK inhibitors SB202190 or SB203580 reduced the alpha-lipoic acid-induced stimulation of glucose uptake by 66 and 55%, respectively. In contrast, SB202474, a structural analog that does not inhibit p38 MAPK, was without effect on glucose uptake. In contrast to 2-deoxyglucose uptake, translocation of GLUT4myc to the cell surface by either alpha-lipoic acid or insulin was unaffected by 20 micromol/l of SB202190 or SB203580. The results suggest that inhibition of 2-deoxyglucose uptake in response to alpha-lipoic acid by inhibitors of p38 MAPK is independent of an effect on GLUT4 translocation. Instead, it is likely that regulation of transporter activity is sensitive to these inhibitors.

Activation of p38 mitogen-activated protein kinase alpha and beta by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport.

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPKalpha and p38 MAPKbeta isoforms were found to be activated (2.1- and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPKalpha and p38 MAPKbeta. The activity of both isoforms was inhibited in vitro by 10 micromol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin- or contraction-stimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 micromol/l SB203580. These results show that p38 MAPKalpha and -beta isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.

Engagement of the insulin-sensitive pathway in the stimulation of glucose transport by alpha-lipoic acid in 3T3-L1 adipocytes.

AIMS/HYPOTHESIS: A natural cofactor of mitochondrial dehydrogenase complexes and a potent antioxidant, alpha-lipoic acid improves glucose metabolism in people with Type II (non-insulin-dependent) diabetes mellitus and in animal models of diabetes. In this study we investigated the cellular mechanism of action of alpha-lipoic acid in 3T3-L1 adipocytes. METHODS: We treated 3T3-L1 adipocytes with 2.5 mmol/l R (+) alpha-lipoic acid for 2 to 60 min, followed by assays of: 2-deoxyglucose uptake; glucose transporter 1 and 4 (GLUT1 and GLUT4) subcellular localization; tyrosine phosphorylation of the insulin receptor or of the insulin receptor substrate-1 in cell lysates; association of phosphatidylinositol 3-kinase activity with immunoprecipitates of proteins containing phosphotyrosine or of insulin receptor substrate-1 using a in vitro kinase assay; association of the p85 subunit of phosphatidylinositol 3-kinase with phosphotyrosine proteins or with insulin receptor substrate-1; and in vitro activity of immunoprecipitated Akt1. The effect of R (+) alpha-lipoic acid was also compared with that of S(-) alpha-lipoic acid. RESULTS: Short-term treatment of 3T3-L1 adipocytes with R (+) alpha-lipoic acid rapidly stimulated glucose uptake in a wortmannin-sensitive manner, induced a redistribution of GLUT1 and GLUT4 to the plasma membrane, caused tyrosine phosphorylation of insulin receptor substrate-1 and of the insulin receptor, increased the antiphosphotyrosine-associated and insulin receptor substrate-1 associated phosphatidylinositol 3-kinase activity and stimulated Akt activity. CONCLUSION/INTERPRETATION: These results indicate that R (+) alpha-lipoic acid directly activates lipid, tyrosine and serine/threonine kinases in target cells, which could lead to the stimulation of glucose uptake induced by this natural cofactor. These properties are unique among all agents currently used to lower glycaemia in animals and humans with diabetes.

Identification of glucagon-like peptide-2 (GLP-2)-activated signaling pathways in baby hamster kidney fibroblasts expressing the rat GLP-2 receptor.

Glucagon-like peptide-2 (GLP-2) promotes the expansion of the intestinal epithelium through stimulation of the GLP-2 receptor, a recently identified member of the glucagon-secretin G protein-coupled receptor superfamily. Although activation of G protein-coupled receptors may lead to stimulation of cell growth, the mechanisms transducing the GLP-2 signal to mitogenic proliferation remain unknown. We now report studies of GLP-2R signaling in baby hamster kidney (BHK) cells expressing a transfected rat GLP-2 receptor (BHK-GLP-2R cells). GLP-2, but not glucagon or GLP-1, increased the levels of cAMP and activated both cAMP-response element- and AP-1-dependent transcriptional activity in a dose-dependent manner. The activation of AP-1-luciferase activity was protein kinase A (PKA) -dependent and markedly diminished in the presence of a dominant negative inhibitor of PKA. Although GLP-2 stimulated the expression of c-fos, c-jun, junB, and zif268, and transiently increased p70 S6 kinase in quiescent BHK-GLP-2R cells, GLP-2 also inhibited extracellular signal-regulated kinase 1/2 and reduced serum-stimulated Elk-1 activity. Furthermore, no rise in intracellular calcium was observed following GLP-2 exposure in BHK-GLP-2R cells. Although GLP-2 stimulated both cAMP accumulation and cell proliferation, 8-bromo-cyclic AMP alone did not promote cell proliferation. These findings suggest that the GLP-2R may be coupled to activation of mitogenic signaling in heterologous cell types independent of PKA via as yet unidentified downstream mediators of GLP-2 action in vivo.

Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts.

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes.

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.

Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC.

2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative chain from ATP production, preventing oxidative metabolism. The consequent increase in energy demand is, however, contested by cells increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal muscle cells, DNP rapidly doubles glucose transport, reminiscent of the effect of insulin. However, glucose transport stimulation by DNP does not require insulin receptor substrate-1 phosphorylation and is wortmannin insensitive. We report here that, unlike insulin, DNP does not activate phosphatidylinositol 3-kinase, protein kinase B/Akt, or p70 S6 kinase. However, chelation of intra- and extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose uptake by 78.9 +/- 3.5%. Because Ca2+-sensitive, conventional protein kinase C (cPKC) can activate glucose transport in L6 muscle cells, we examined whether cPKC may be translocated and activated in response to DNP in L6 myotubes. Acute DNP treatment led to translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from plasma membranes exhibited a twofold increase in kinase activity in response to DNP. Overnight treatment with 4-phorbol 12-myristate 13-acetate downregulated cPKC isoforms alpha, beta, and gamma and partially inhibited (45.0 +/- 3.6%) DNP- but not insulin-stimulated glucose uptake. Consistent with this, the PKC inhibitor bisindolylmaleimide I blocked PKC enzyme activity at the plasma membrane (100%) and inhibited DNP-stimulated 2-[3H]deoxyglucose uptake (61.2 +/- 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC-beta inhibitor LY-379196 partially inhibited DNP effects on glucose uptake (66.7 +/- 1.6%). The results suggest interfering with mitochondrial ATP production acts on a signal transduction pathway independent from that of insulin and partly mediated by Ca2+ and cPKCs, of which PKC-beta likely plays a significant role.

Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta.

Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total 86Rb+ (K+) uptake from 0.8 +/- 0.04 to 1.02 +/- 0.05 nmol.mg-1.protein-1.min-1 (p < 0.005). These changes in K+ flux, though small, can alter the membrane potential. Uptake occurred through both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na+/K+/2Cl- cotransporter prior to insulin action, no increase in 86Rb+ uptake via the Na+/K(+)-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50-200 nmol/l) and LY294002 (50 mumol/l) attenuated both total insulin-stimulated 86Rb+ uptake as well as uptake via the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter. Neither the inhibitor of p70.S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098059 (50 mumol/l), had any effect on insulin's stimulation of K+ influx. A 10 mumol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 mumol/l it was ineffective, suggesting involvement of the atypical PKC-zeta isoform. We conclude that insulin-stimulated K+ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-zeta.

Actin filaments facilitate insulin activation of the src and collagen homologous/mitogen-activated protein kinase pathway leading to DNA synthesis and c-fos expression.

The exact mechanism of the spatial organization of the insulin signaling pathway leading to nuclear events remains unknown. Here, we investigated the involvement of the actin cytoskeleton in propagation of insulin signaling events leading to DNA synthesis and expression of the immediate early genes c-fos and c-jun in L6 muscle cells. Insulin reorganized the cellular actin network and increased the rate of DNA synthesis and the levels of c-fos mRNA, but not those of c-jun mRNA, in undifferentiated L6 myoblasts. Similarly, insulin markedly elevated the levels of c-fos mRNA but not of c-jun mRNA in differentiated L6 myotubes. Disassembly of the actin filaments by cytochalasin D, latrunculin B, or botulinum C2 toxin significantly inhibited insulin-mediated DNA synthesis in myoblasts and abolished stimulation of c-fos expression by the hormone in myoblasts and myotubes. Actin disassembly abolished insulin-induced phosphorylation and activation of extracellulor signal-regulated kinases, activation of a 65-kda member of the p21-activated kinases, and phosphorylation of p38 mitogen-activated protein kinases but did not prevent activation of phosphatidylinositol 3-kinase and p70(S6k). Under these conditions, insulin-induced Ras activation was also abolished, and Grb2 association with the Src and collogen homologous (Shc) molecule was inhibited without inhibition of the tyrosine phosphorylation of Shc. We conclude that the actin filament network plays an essential role in insulin regulation of Shc-dependent signaling events governing gene expression by facilitating the interaction of Shc with Grb2.

Temporal activation of p70 S6 kinase and Akt1 by insulin: PI 3-kinase-dependent and -independent mechanisms.

Several studies have suggested that activation of p70 ribosomal S6 kinase (p70 S6 kinase) by insulin may be mediated by the phosphatidylinositol 3-kinase (PI 3-kinase)-Akt pathway. However, by temporal analysis of the activation of each kinase in L6 muscle cells, we report that the activation of the two serine/threonine kinases (Akt and p70 S6 kinase) can be dissociated. Insulin stimulated p70 S6 kinase in intact cells in two phases. The first phase (5 min) of stimulation was fully inhibited by wortmannin (IC50 = 20 nM) and LY-294002 (full inhibition at 5 microM). After this early inhibition, p70 S6 kinase was gradually stimulated by insulin in the presence of 100 nM wortmannin. After 30 min, the stimulation was 65% of the maximum attained in the absence of wortmannin. The IC50 of wortmannin for inhibition of this second phase was approximately 150 nM. In contrast, activation of Akt1 by insulin was completely inhibited by 100 nM wortmannin at all time points investigated. Inhibition of mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase with PD-098059 (10 microM) or treatment with the protein kinase C inhibitor bisindolylmaleimide (10 microM) had no effect on the late phase of insulin stimulation of p70 S6 kinase. We have previously shown that GLUT-1 protein synthesis in these cells is stimulated by insulin via the mTOR-p70 S6 kinase pathway, based on its sensitivity to rapamycin. We therefore investigated whether the signals leading to GLUT-1 synthesis correlated with the early or late phase of stimulation of p70 S6 kinase. GLUT-1 synthesis was not inhibited by wortmannin (100 nM). In summary, insulin activates p70 ribosomal S6 kinase in L6 muscle cells by two mechanisms, one dependent on and one independent of the activation of PI 3-kinase. In addition, activation of Akt1 is fully inhibited by wortmannin, suggesting that Akt1 does not participate in the late activation of p70 S6 kinase. Wortmannin-sensitive PI 3-kinases and Akt1 are not required for insulin stimulation of GLUT-1 protein biosynthesis.

Dexamethasone stimulates the expression of GLUT1 and GLUT4 proteins via different signalling pathways in L6 skeletal muscle cells.

It was recently demonstrated that dexamethasone treatment of L6 skeletal muscle cells resulted in an elevation of GLUT1 protein. However, the level of GLUT4 protein under these conditions was not examined. In addition, the signalling mechanism(s) leading to dexamethasone-induced expression of GLUT1 protein was not investigated. In the present study we investigated the effect of dexamethasone on the expression of GLUT1 and GLUT4 proteins in differentiated L6 muscle cells and the signalling mechanism(s) via which dexamethasone may act. Dexamethasone (300 nM) treatment for 24 h elevated GLUT1 and GLUT4 proteins by 68% and 94%, respectively, above control levels. These increases were due to de novo synthesis as shown by metabolic labelling with [35S]methionine. Incubation of cells with 100 nM wortmannin or 30 ng/ml rapamycin prevented the dexamethasone-stimulated elevation of GLUT1 protein. In contrast, neither of these inhibitors affected the elevation of GLUT4 protein by dexamethasone. Furthermore, dexamethasone down-regulated insulin receptor substrate-1 protein content by 42% and insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 by 28%. The p70 ribosomal S6 kinase was not activated by dexamethasone and instead, dexamethasone attenuated the stimulation of this enzyme activity by insulin. These results suggest that dexamethasone induces the expression of GLUT1 and GLUT4 protein by independent signalling mechanisms with a concomitant depression of intracellular signalling by insulin.

Stimulation of glucose and amino acid transport and activation of the insulin signaling pathways by insulin lispro in L6 skeletal muscle cells.

The monomeric insulin analogue insulin lispro (Lys B28, Pro B29) is a rapid-acting insulin with a shorter duration of activity than human regular insulin. This compound has the advantage of reducing early postprandial hyperglycemia and the accompanying late hypoglycemia, thereby improving overall blood glucose control. To date, all published studies of the functional properties of insulin lispro have been conducted in whole animals. This study aimed to characterize the cellular actions of insulin lispro and the signals it elicits in an insulin-sensitive muscle cell line, L6 cells. Comparing the cellular actions of insulin lispro with those of human regular insulin, a number of observations were made. (1) Insulin lispro stimulated glucose and amino acid transport into L6 myotubes with a dose dependency and time course virtually identical to those of human regular insulin. (2) Insulin lispro was as effective as human regular insulin in stimulating time-dependent phosphorylation of insulin receptor substrate 1 (IRS-1), p70 ribosomal S6 kinase, and two isoforms of mitogen-activated protein kinase (ERK1 and ERK2). (3) Insulin lispro's ability to induce the association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase was similar to that of human regular insulin. (4) As with human regular insulin, 100 nmol of the fungal metabolite wortmannin completely inhibited insulin lispro stimulation of glucose uptake. We concluded that the cellular actions of insulin lispro are similar to those of human regular insulin with respect to glucose and amino acid uptake and that the biochemical signals elicited are also comparable.

Dexamethasone stimulates the expression of GLUT1 and GLUT4 proteins via different signalling pathways in L6 skeletal muscle cells.

It was recently demonstrated that dexamethasone treatment of L6 skeletal muscle cells resulted in an elevation of GLUT1 protein. However, the level of GLUT4 protein under these conditions was not examined. In addition, the signalling mechanism(s) leading to dexamethasone-induced expression of GLUT1 protein was not investigated. In the present study we investigated the effect of dexamethasone on the expression of GLUT1 and GLUT4 proteins in differentiated L6 muscle cells and the signalling mechanism(s) via which dexamethasone may act. Dexamethasone (300 nM) treatment for 24 h elevated GLUT1 and GLUT4 proteins by 68% and 94%, respectively, above control levels. These increases were due to de novo synthesis as shown by metabolic labelling with [35S]methionine. Incubation of cells with 100 nM wortmannin or 30 ng/ml rapamycin prevented the dexamethasone-stimulated elevation of GLUT1 protein. In contrast, neither of these inhibitors affected the elevation of GLUT4 protein by dexamethasone. Furthermore, dexamethasone down-regulated insulin receptor substrate-1 protein content by 42% and insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 by 28%. The p70 ribosomal S6 kinase was not activated by dexamethasone and instead, dexamethasone attenuated the stimulation of this enzyme activity by insulin. These results suggest that dexamethasone induces the expression of GLUT1 and GLUT4 protein by independent signalling mechanisms with a concomitant depression of intracellular signalling by insulin.

Insulin regulation and selective segregation with glucose transporter-4 of the membrane aminopeptidase vp165 in rat skeletal muscle cells.

vp165, a recently described member of the family of zinc-dependent membrane aminopeptidases, is a major constituent of glucose transporter-4 (GLUT4)-containing vesicles in adipocytes and skeletal muscle. Here we show that vp165 is expressed in L6 myoblasts and increases by 4.3-fold during differentiation into myotubes. The localization of vp165 in L6 myotubes was assessed by immunoblotting subcellular fractions from basal and insulin-stimulated cells and was compared to the distribution of GLUT4. vp165 and GLUT4 were mainly concentrated in the low density microsomal membranes under basal conditions. Upon stimulation with insulin, vp165 and GLUT4 were redistributed from the low density microsomes to the plasma membrane. The majority of vp165 was found in immunoisolated GLUT4-containing vesicles, and vice versa, the majority of GLUT4 was detected in immunoisolated vp165-containing vesicles. In contrast, the two other glucose transporter isoforms expressed in L6, GLUT1 and GLUT3, were excluded from GLUT4- and vp165-containing vesicles. These results suggest that in rat skeletal muscle cells, vp165 and GLUT4 cosegregate to the same intracellular compartment and that this is distinct from the compartment containing GLUT1 and GLUT3.