Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK.

BACKGROUND: We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR). METHODS: Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit(-/-) embryos (AMPKα1/2(-/-)-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC). RESULTS: Metformin (2.5 µM-5 mM) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)-AMPK-p53/p21(cip1) and inhibited the Akt-mammalian target of rapamycin (mTOR)-eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2(-/-)-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM-AMPK-p53/p21(cip1) and inhibition of Akt-mTOR-4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers. CONCLUSION: Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM-AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC.

Stimulation of glucose uptake by the natural coenzyme alpha-lipoic acid/thioctic acid: participation of elements of the insulin signaling pathway.

Thioctic acid (alpha-lipoic acid), a natural cofactor in dehydrogenase complexes, is used in Germany in the treatment of symptoms of diabetic neuropathy. Thioctic acid improves insulin-responsive glucose utilization in rat muscle preparations and during insulin clamp studies performed in diabetic individuals. The aim of this study was to determine the direct effect of thioctic acid on glucose uptake and glucose transporters. In L6 muscle cells and 3T3-L1 adipocytes in culture, glucose uptake was rapidly increased by (R)-thioctic acid. The increment was higher than that elicited by the (S)-isomer or the racemic mixture and was comparable with that caused by insulin. In parallel to insulin action, the stimulation of glucose uptake by thioctic acid was abolished by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, in both cell lines. Thioctic acid provoked an upward shift of the glucose-uptake insulin dose-response curve. The molar content of GLUT1 and GLUT4 transporters was measured in both cell lines. 3T3-L1 adipocytes were shown to have >10 times more glucose transporters but similar ratios of GLUT4:GLUT1 than L6 myotubes. The effect of (R)-thioctic acid on glucose transporters was studied in the L6 myotubes. Its stimulatory effect on glucose uptake was associated with an intracellular redistribution of GLUT1 and GLUT4 glucose transporters, similar to that caused by insulin, with minimal effects on GLUT3 transporters. In conclusion, thioctic acid stimulates basal glucose transport and has a positive effect on insulin-stimulated glucose uptake. The stimulatory effect is dependent on phosphatidylinositol 3-kinase activity and may be explained by a redistribution of glucose transporters. This is evidence that a physiologically relevant compound can stimulate glucose transport via the insulin signaling pathway.

Multiple roles of phosphatidylinositol 3-kinase in regulation of glucose transport, amino acid transport, and glucose transporters in L6 skeletal muscle cells.

Phosphatidylinositol 3-kinase (PI3k) activity is required for the insulin stimulation of glucose transport in adipocytes and Chinese hamster ovary cells. Wortmannin (WM), an inhibitor of PI3k, inhibits the stimulation of glucose transport by insulin and the gain of glucose transporters at the cell surface. However, the effect of inhibition of PI3k on the maintenance of the basal and the insulin-stimulated glucose transport and on the intracellular donor pool of glucose transporters has not been clarified. Here we show that in L6 skeletal muscle cells in culture WM significantly inhibits the basal PI3k activity (by 40%), decreases the levels of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate (by about 50%) and abolishes the activation of the enzyme by insulin. WM inhibited the basal rate of transport of glucose (by 45%) and of amino acids through system A (by 25%) and abolished their stimulation by insulin. Insulin caused a transient increase in PI3k activity and PI3k products that returned to basal levels within 40 min, whereas glucose and amino acid transport remained elevated. Under these conditions, WM reduced the rate of glucose and amino acid transport back to basal levels. In unstimulated cells, WM decreased significantly the GLUT4 glucose transporter content at the plasma membrane and prevented the ability of insulin to recruit transporters to this membrane. Interestingly, the intracellular pools of the GLUT3 and GLUT4 glucose transporters were significantly reduced in response to WM treatment alone. We conclude that in muscle cells PI3k activity is required to maintain basal and insulin-stimulated glucose and amino acid transport, as well as to develop the stimulation of the two transport processes in response to the hormone. We hypothesize that PI3k, likely through production of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate, regulates the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool, in addition to being an insulin signal.

Role of the actin cytoskeleton in insulin action.

Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, 1) adaptor molecules such as the insulin receptor substrates (IRS), the Src and collagen homologs (Shc), and the growth factor receptor binding protein 2 (Grb2); 2) lipid kinases such as phosphatidylinositol 3-kinase (PI 3-Kinase); 3) small G proteins; and 4) serine, threonine, and tyrosine kinases. The activation of such signaling molecules by insulin is now well established, but we do not yet fully understand the mechanisms integrating these seemingly diverse pathways. Here, we discuss the involvement of the actin cytoskeleton in the propagation and regulation of insulin signals. In muscle cells in culture, insulin induces a rapid actin filament reorganization that coincides with plasma membrane ruffling and intense accumulation of pinocytotic vesicles. Initiation of these effects of insulin requires an intact actin cytoskeleton and activation of PI 3-kinase. We observed recruitment PI 3-kinase subunits and glucose transporter proteins to regions of reorganized actin. In both muscle and adipose cells, actin disassembly inhibited early insulin-induced events such as recruitment of glucose transporters to the cell surface and enhanced glucose transport. Additionally, actin disassembly inhibited more prolonged effects of insulin, including DNA synthesis and expression of immediate early genes such as c-fos. Intact actin filaments appear to be essential for mediation of early events such as association of Shc with Grb2 in response to insulin, which leads to stimulation of gene expression. Preliminary observations support a role for focal adhesion signaling complexes in insulin action. These observations suggest that the actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating proper subcellular distribution of signaling molecules that participate in the insulin signaling pathway.

Exercise increases the plasma membrane content of the Na+ -K+ pump and its mRNA in rat skeletal muscles.

Muscle fibers adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exercise training has been shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercise has not been determined, and it is not known whether it involves alterations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of treadmill running (20 m/min, 10% grade) on the subcellular distribution and expression of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and IIb (white-IIa/IIb) hindlimb muscles from resting and exercised female Sprague-Dawley rats were removed for subcellular fractionation. By homogenization and gradient centrifugation, crude membranes and purified plasma membranes were isolated and subjected to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthermore, mRNA was isolated from specific red type I (red-I) and white type IIb (white-IIb) muscles and subjected to Northern blotting by using subunit-specific probes. In both red-I/IIa and white-IIa/IIb muscles, exercise significantly raised the plasma membrane content of the alpha1-subunit of the pump by 64 +/- 24 and 55 +/- 22%, respectively (P < 0.05), and elevated the alpha2-polypeptide by 43 +/- 22 and 94 +/- 39%, respectively (P < 0.05). No significant effect of exercise could be detected on the amount of these subunits in an internal membrane fraction or in total membranes. In addition, exercise significantly increased the alpha1-subunit mRNA in red-I muscle (by 50 +/- 7%; P < 0.05) and the beta2-subunit mRNA in white-IIb muscles (by 64 +/- 19%; P < 0.01), but the alpha2- and beta1-mRNA levels were unaffected in this time period. We conclude that increased presence of alpha1- and alpha2-polypeptides at the plasma membrane and subsequent elevation of the alpha1- and beta2-subunit mRNAs may be mechanisms by which acute exercise regulates the Na+-K+ pump of skeletal muscle.

Predicting 2-year survival for radiation regimens in advanced non-small cell lung cancer.

AIMS: Total dose, dose per fraction, number of fractions and treatment time are important determinants of the biological effect of a radiation regimen. Several randomised clinical trials (RCTs) have tested a variety of dosing regimens in advanced unresected non-small cell lung cancer, but survival remains poor. This work used past RCT data to develop and validate a predictive model that could help in designing new radiation regimens for successful testing in RCTs. MATERIALS AND METHODS: Eleven RCTs that compared radiation regimens alone were used to define the relationship between radiation regimens and 2-year survival. On the basis of this relationship, predictive models were developed. Predicted values were internally and externally validated against observed values from the same 11 RCTs and 21 other RCTs. Scatter plots and Pearson's correlation coefficient (r) were used for validation. Finally, regimens were explored that could improve survival. RESULTS: Increments in the total dose, dose per day and the number of treatment days were associated with improved survival; increments in dose-squared and treatment weeks were associated with reduced survival. The observed and predicted values were similar on internal (r = 0.96) and external validation (r = 0.76). Regimens that delivered a higher total dose over a shorter time had higher survival rates compared with the standard (60 Gy, 30 fractions, 6 weeks); survival may be improved by delivering the standard treatment in 5 weeks rather than 6 weeks. CONCLUSION: The developed model can predict the effect of thoracic radiation on survival in advanced non-small cell lung cancer patients. It is a useful tool for designing new radiation regimens for clinical trials.

Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action.

In L6 myotubes insulin stimulates glucose transport through the translocation of glucose transporters GLUT1, GLUT3 and GLUT4 from intracellular stores to the plasma membrane. An intact actin network and phosphatidylinositol 3-kinase activity are required for this process. Glucose transport is also stimulated by the mitochondrial ATP-production uncoupler dinitrophenol. We show here that, in serum-depleted myotubes, dinitrophenol induced translocation of GLUT1 and GLUT4, but not GLUT3. This response was not affected by inhibiting phosphatidylinositol 3-kinase or disassembling the actin network. Insulin, but not dinitrophenol, caused tyrosine phosphorylation of several polypeptides, including the insulin-receptor substrate-1 and mitogen-activated protein kinase. Similarly, insulin, but not dinitrophenol, caused actin reorganization, which was inhibited by wortmannin. We conclude that insulin and dinitrophenol stimulate glucose transport by different mechanisms.

Disassembly of the actin network inhibits insulin-dependent stimulation of glucose transport and prevents recruitment of glucose transporters to the plasma membrane.

In muscle and fat tissues, insulin stimulates glucose transport through the translocation of glucose transporter proteins from an intracellular storage pool to the plasma membrane. The mechanism of this translocation is unknown. We have examined the possible role of the actin microfilament network in the stimulation of glucose transport by insulin and on the distribution of glucose transporters, in differentiated L6 rat skeletal muscle cells. Insulin (10(-7) M for 30 min) caused a major reorganization of the actin network of differentiated L6 myotubes. Cytochalasin D, a widely used inhibitor of actin filament formation, caused a dose- and time-dependent disassembly of the actin network, which was associated with an 80% inhibition of the insulin stimulation of glucose transport, without affecting the basal rate of glucose uptake. L6 myotubes express three glucose transporter isoforms, named GLUT1, GLUT3, and GLUT4. Disassembly of the actin network by cytochalasin D did not affect the number of basal glucose transporters in the plasma membrane but reduced the content of all three glucose transporters in intracellular membranes and prevented their appearance at the plasma membrane response to insulin. The inhibitory effect of cytochalasin D treatment on the insulin stimulation of glucose transport occurred downstream of tyrosine phosphorylation of the insulin receptor substrate-1 and of binding of phosphatidylinositol 3-kinase to the insulin receptor substrate-1. Using immunoprecipitation of intact membranes, we detected specific association of the actin-binding protein spectrin with GLUT4 glucose transporter-containing vesicles. We conclude that an intact actin network is required for the correct intracellular localization of glucose transporters, as well as for their incorporation into the plasma membrane in response to insulin. A direct interaction may exist between the actin network and the glucose transporter vesicles which may be mediated through a spectrin-containing skeleton attached to glucose transporter-containing vesicles.

Glucose transporter expression in L6 muscle cells: regulation through insulin- and stress-activated pathways.

We addressed the effect of long-term treatment with insulin, 2,4-dinitrophenol (DNP; an uncoupler of oxidative phosphorylation that increases energy demand) and 300 mM mannitol (hyperosmolarity) on glucose transporter (GLUT) expression in L6 muscle cells and the signaling pathways involved. We found the following. 1) The insulin-mediated increase in GLUT-1 is 70-kDa ribosomal protein S6 kinase (p70 S6 kinase) and p38 mitogen-activated protein kinase (MAPK) dependent but extracellular signal-regulated protein kinase (ERK) and MAPK/ERK kinase (MEK) independent. The hypertonicity-stimulated elevation in GLUT-1 is p70 S6 kinase, p38 MAPK, and MEK dependent yet ERK independent. DNP also increased GLUT-1 protein but did not depend on any of the above pathways, 2) Insulin increased GLUT-3 protein in a p70 S6 kinase-independent but MEK/ERK-dependent fashion. Inhibition of p38 MAPK potentiated the effect of insulin on GLUT-3. Hypertonicity increased GLUT-3 via p70 S6 kinase- and p38 MAPK-dependent pathways. In conclusion, we have dissected the molecular mechanisms used by insulin and hypertonicity that culminate in the induction of GLUT-1 and GLUT-3. The mechanism(s) used by DNP remains unknown.

Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures.

Glucose transporters are membrane-embedded proteins that mediate the uptake of glucose from the surrounding medium into the cell. Glucose is the main fuel for most cells, and its uptake is rate-limiting for glucose utilization. For this reason, it is expected that glucose transport is tightly regulated. Whereas rapid regulation of glucose transporters by hormones has been known for some time, the regulation of glucose transporters by substrate availability (i.e., by glucose itself) is less well understood. This question has been approached by scientists from two angles: one, by measuring the consequence of diabetic states (in which there is surplus of glucose availability) on the expression of glucose transporter genes, and another one, by measuring the effect of glucose availability and glucose deprivation in cell cultures on glucose transporter gene expression. The results from both camps are unfortunately not coincident, due in part to the coexistence of other variables in the diabetic animals, and to the lack of ideal cell cultures. In spite of these caveats, the profuse literature on both approaches propelled us to find commonalities within each approach. This review concludes that in animal studies, one isoform of glucose transporters, the GLUT4 type, is down-regulated by high levels of circulating glucose in muscle but not in fat cells. This down-regulation of the protein is independent of regulation of transcription. In contrast, in fat cells, high glucose levels depress GLUT4 mRNA levels. In cell culture studies, high glucose levels lead to lower expression of the GLUT1 transporter isoform relative to glucose-deprived cultures. Glucose levels do not affect the amount of GLUT4 transporter isoform. The down-regulation of the GLUT1 transporter protein is caused by pre- and post-transcriptional mechanisms, the prevalence of each being cell-type specific. No glucose-responsive elements have been identified on either the GLUT1 or GLUT4 genes, and no information is available on the glucose metabolites that mediate the response of glucose transporter gene expression to glucose availability.

Insulin activates a p21-activated kinase in muscle cells via phosphatidylinositol 3-kinase.

Insulin activates rapidly a complex cascade of lipid and protein kinases leading to stimulation of mitogenic and metabolic events. Here we describe a renaturable kinase of 65 kDa (PK65) that becomes rapidly activated by insulin in differentiated L6 muscle cells (myotubes) and can phosphorylate histones immobilized in polyacrylamide gels. Insulin activation of PK65 was abolished by the tyrosine kinase inhibitor erbstatin and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, but was unaffected by inhibitors of protein kinase C or of the activation of p70(S6K). Recently, a number of protein kinases have been described which become activated through interaction with the small GTP-binding proteins Rac and Cdc42 (21-ctivated inases, or PAKs) and lead to activation of the stress-induced mitogen-activated protein kinase (MAPK) p38 MAPK. Two different polyclonal antibodies recognizing the carboxyl-terminal or the Rac-binding domain of a 65-kDa PAK (PAK65) immunoprecipitated the myotube PK65. The insulin-induced activation of PK65 in myotubes was detectable following immunoprecipitation of the kinase. Furthermore, PK65 associated with and became activated by glutathione S-transferase-Cdc42Hs in the presence of GTPgammaS (guanosine 5'-3-O-(thio)triphosphate). In myotubes insulin also induced tyrosine phosphorylation of p38 MAPK. However, this phosphorylation was insensitive to wortmannin, indicating that p38 MAPK is not activated by PK65 in insulin-stimulated cells. The results suggest that insulin activates in muscle cells a renaturable kinase (PK65) closely related to PAK65. Tyrosine kinases and PI 3-kinase act upstream of PK65 in the insulin signaling cascade. Insulin activates p38 MAPK in myotubes, but this occurs by a pathway independent of PI 3-kinase and PK65.

Involvement of the actin network in insulin signalling.

The purpose of the studies included in this chapter was to examine the role of the actin network in the propagation of insulin action leading to stimulation of glucose transport and activation of the mitogen-activated protein kinase cascade. The active insulin receptor phosphorylates tyrosine residues of intracellular proteins such as the insulin receptor substrate-1 (IRS-1) which acts as docking sites for molecules containing Src homology 2 (SH2) domains. One such molecule is phosphatidylinositol 3-kinase (PI 3-kinase) which becomes activated by binding to IRS-1. PI 3-kinase activity is required for the insulin-stimulation of glucose transport and glycogen synthesis. Grb2, a small adaptor molecule, can bind IRS-1 and, through the guanine nucleotide exchange factor Sos, leads to the activation of the small GTP binding protein Ras. Through a cascade of protein kinases, activation of Ras results in activation of the Erk 1 and 2 mitogen-activated protein kinases (MAPKs) which appear to control important nuclear and metabolic events. To investigate the role of the actin network in the propagation of insulin action leading to stimulation of glucose transport and the activation of the Erk MAPKs, we used the fungal metabolite cytochalasin D which disassembles the actin network. Actin disassembly abolished almost completely the ability of insulin to increase the rate of glucose transport into L6 muscle cells (myotubes) through prevention of the insulin-induced recruitment of glucose transporters to the plasma membrane which is the event that mediates the increase in the rate of transport. Actin disassembly did not affect either the insulin-mediated phosphorylation of IRS-1, the association of PI 3-kinase with this molecule, or the activation of IRS-1-associated PI 3-kinase. These results were also verified in another insulin responsive cell line, the 3T3-L1 adipocytes. In these cells, actin disassembly inhibited the insulin-induced recruitment of PI 3-kinase to intracellular membranes containing glucose transporters. Moreover, actin disassembly abolished the insulin-mediated phosphorylation of the Erk MAPKs. We conclude that the cellular actin network of insulin responsive cells is not required for the activation of PI 3-kinase but prevents its cellular redistribution. In contrast, intact actin filaments are essential for the propagation of insulin signals leading to the the activation of the MAPKs.

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.

Structural disruption of the trans-Golgi network does not interfere with the acute stimulation of glucose and amino acid uptake by insulin-like growth factor I in muscle cells.

The effects of insulin-like growth factor I (IGF-I) on glucose and amino acid uptake were investigated in fully differentiated L6 muscle cells, in order to determine whether the two processes are functionally related. Transport of both glucose and amino acid (methylaminoisobutyric acid, MeAIB) was activated rapidly in response to IGF-I. Stimulation reached a peak within 30 min and was sustained for up to 90 min. Maximal activation of either glucose or MeAIB transport was achieved at 3 nM IGF-I; the half-maximal activation (ED50) of glucose transport was at 107 pM and that of MeAIB transport was at 36 pM. Stimulation of amino acid uptake occurred in the absence or presence of glucose, suggesting that this response is not secondary to increased glucose intake. Incubation of cells for 1 h with Brefeldin A (5 micrograms/ml), which disassembles the Golgi apparatus and inhibits the secretory pathway in eukaryotic cells, had no effect on the acute IGF-I activation of glucose and MeAIB transport. Moreover, Brefeldin A caused wide redistribution of the trans-Golgi antigen TGN38, as assessed by subcellular fractionation, without affecting the distribution of glucose transporters. The finding that the degree of activation, time response and sensitivity to IGF-I and Brefeldin A were similar for both glucose and MeAIB transport suggests commonalities in the IGF-I mechanism of recruitment of glucose transporters and stimulation of amino acid transport through System A. An integral trans-Golgi network does not appear to be required for the acute IGF-I stimulation of glucose or amino acid transport, even though stimulation of glucose transport occurs through recruitment of glucose transporters from intracellular stores in these cells. We propose that the donor site of glucose transporters (and perhaps of amino acid transporters) involved in the acute response to IGF-I lies beyond the trans-Golgi network, perhaps in an endosomal compartment in close proximity to the plasma membrane.

Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes.

Insulin stimulates the rate of glucose uptake into muscle and adipose cells by translocation of glucose transporters from an intracellular storage pool to the plasma membrane. This event requires the prior activation of phosphatidylinositol 3-kinase (PI 3-kinase). Here we report that insulin causes an increase in wortmannin-sensitive PI 3-kinase activity and a gain in the enzyme's regulatory and catalytic subunits p85alpha and p110beta (but not p110alpha) in the intracellular compartments containing glucose transporters. The hormone also caused a marked reorganization of actin filaments, which was prevented by cytochalasin D. Cytochalasin D also decreased significantly the insulin-dependent association of PI 3-kinase activity and the levels of insulin receptor substrate (IRS)-1, p85alpha and p110beta with immunopurified GLUT4-containing compartments. In contrast, the drug did not alter the insulin-induced tyrosine phosphorylation of IRS-1, the association of PI 3-kinase with IRS-1, or the stimulation of PI 3-kinase by insulin in anti-(IRS-1) or anti-p85 immunoprecipitates from whole cell lysates. Cytochalasin D, and the chemically unrelated latrunculin B, which also inhibits actin filament reassembly, prevented the insulin stimulation of glucose transport by approx. 50%. Cytochalasin D decreased by about one-half the insulin-dependent translocation to the plasma membrane of the GLUT1 and GLUT4 glucose transporters. The results suggest that the existence of intact actin filament is correlated with the full recruitment of glucose transporters by insulin. The underlying function of the actin filaments might be to facilitate the insulin-mediated association of the p85-p110 PI 3-kinase with glucose-transporter-containing compartments.

Insulin, insulin-like growth factor-I, and platelet-derived growth factor activate extracellular signal-regulated kinase by distinct pathways in muscle cells.

We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.

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.