Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK beta-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.     

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.     

Effect of inhibiting vacuolar acidification on insulin signaling in hepatocytes

Previous studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction.     

Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator

System A, the Na(+)-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.     

Amino acid transporter ATA2 is stored at the trans-Golgi network and released by insulin stimulus in adipocytes

Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of alpha-(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-d-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.     

Insulin receptor internalization and signalling

The insulin receptor kinase (IRK) is a tyrosine kinase whose activation, subsequent to insulin binding, is essential for insulin-signalling in target tissues. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-IRK complexes into the endosomal apparatus (EN) of the cell. Internalization of insulin into target organs, especially liver, is implicated in effecting insulin clearance from the circulation. Internalization mediates IRK downregulation and hence attenuation of insulin sensitivity although most internalized IRKs readily recycle to the plasma membrane at physiological levels of insulin. A role for internalization in insulin signalling is indicated by the accumulation of activated IRKs in ENs. Furthermore, the maximal level of IRK activation has been shown to exceed that attained at the cell surface. Using an in vivo rat liver model in which endosomal IRKs are exclusively activated has revealed that IRKs at this intracellular locus are able by themselves to promote IRS-1 tyrosine phosphorylation and induce hypoglycemia. Furthermore, studies with isolated rat adipocytes reveal the EN to be the principle site of insulin-stimulated IRS-1 tyrosine phosphorylation and associated PI3K activation. Key steps in the termination of the insulin signal are also operative in ENs. Thus, an endosomal acidic insulinase has been identified which limits the extent of IRK activation. Furthermore, IRK dephosphorylation is effected in ENs by an intimately associated phosphotyrosine phosphatase(s) which, in rat liver, appears to regulate IRK activity in both a positive and negative fashion. Thus, insulin-mediated internalization of IRKs into ENs plays a crucial role in effecting and regulating signal transduction in addition to modulating the levels of circulating insulin and the cellular concentration of IRK in target tissues.     

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p) . Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.     

Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.     

The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway

Phosphoinositide-3 kinase (PI-3 kinase) and its downstream signaling molecules PDK-1 and Akt were analyzed in SK-N-SH and SK-N-BE(2) human neuroblastoma cell lines. When cells were stimulated with insulin, PI-3 kinase was activated in both cell lines, whereas the translocation of PDK-1 to the membrane fraction and phosphorylated Akt were observed only in SK-N-SH cells. Analyses of the insulin-mediated reactive oxygen species (ROS) generation and Phosphatase and Tensin homolog (PTEN) oxidation indicate that PTEN oxidation occurred in SK-N-SH cells, which can produce ROS, but not in SK-N-BE(2) cells, which cannot increase ROS in response to insulin stimulation. When SK-N-SH cells were pretreated with the NADPH oxidase inhibitor diphenyleneiodonium chloride before insulin stimulation, insulin-mediated translocation of PDK-1 to the membrane fraction and phosphorylation of Akt were remarkably reduced, whereas PI-3 kinase activity was not changed significantly. These results indicate that not only PI-3 kinase activation but also inhibition of PTEN by ROS is needed to increase cellular level of phosphatidylinositol 3,4,5-trisphosphate for recruiting downstream signaling molecules such as PDK-1 and Akt in insulin-mediated signaling. Moreover, the ROS generated by insulin stimulation mainly contributes to the inactivation of PTEN and not to the activation of PI-3 kinase in the PI-3 kinase/Akt pathway.     

Rab4b Is a Small GTPase Involved in the Control of the Glucose Transporter GLUT4 Localization in Adipocyte

Background

Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport.

Methodology/Principal Findings

We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment.

Conclusion/Significance

Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes.     

Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells

Amino acids have emerged as potent modulators of the mTOR/p70 S6 kinase pathway. The involvement of this pathway in the regulation of insulin-stimulated glucose transport was investigated in the present study. Acute exposure (1 h) to a balanced mixture of amino acids reduced insulin-stimulated glucose transport by as much as 55% in L6 muscle cells. The effect of amino acids was fully prevented by the specific mTOR inhibitor rapamycin. Time course analysis of insulin receptor substrate 1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity revealed that incubation with amino acids speeds up its time-dependent deactivation, leading to a dramatic suppression (-70%) of its activity after 30 min of insulin stimulation as compared with its maximal activation (5 min of stimulation). This accelerated deactivation of PI 3-kinase activity in amino acid-treated cells was associated with a concomitant and sustained increase in the phosphorylation of p70 S6 kinase. In marked contrast, inhibition of mTOR by rapamycin maintained PI 3-kinase maximally activated for up to 30 min. The marked inhibition of insulin-mediated PI 3-kinase activity by amino acids was linked to a rapamycin-sensitive increase in serine/threonine phosphorylation of IRS-1 and a decreased binding of the p85 subunit of PI 3-kinase to IRS-1. Furthermore, amino acids were required for the degradation of IRS-1 during long term insulin treatment. These results identify the mTOR/p70 S6 kinase signaling pathway as a novel modulator of insulin-stimulated glucose transport in skeletal muscle cells.     

Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.     

The serine/threonine kinases SGK1, 3 and PKB stimulate the amino acid transporter ASCT2

The human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2/SLC1A5) plays an important role in the transport of neutral amino acids in epithelial cells. The serine and threonine kinases SGK1-3 and protein kinase B have been implicated in the regulation of several members of the SLC1 transporter family by enhancing their plasma membrane abundance. The present study explored whether those kinases modulate hASCT2. In Xenopus oocytes heterologously expressing hASCT2, coexpression of constitutively active (S422D)SGK1, (S419D)SGK3 or (T308DS473D)PKB upregulated the transporter activity. The stimulation requires the catalytical activity of the kinases since the inactive mutants (K127N)SGK1, (K191N)SGK3, and (T308AS473A)PKB failed to modulate the transporter. According to kinetic analysis and chemiluminescence assays, SGK1 and SGK3 modulate hASCT2 by enhancing the transporter abundance in the plasma membrane. As SGK1, 3 and PKB are activated by insulin and IGF1, the described mechanisms presumably participate in the hormonal stimulation of cellular amino acid uptake.     

Anterograde and retrograde transport of neutral sphingomyelinase-2 between the Golgi and the plasma membrane

The activation of neutral sphingomyelinase-2 (nSMase2) and consequent ceramide production are implicated in many stress-induced signaling pathways. Trafficking of nSMase2 from the Golgi compartment to the plasma membrane (PM) in response to signaling stimuli has been described. However, the precise mechanisms of transport remain unknown. This study aimed to investigate the trafficking of nSMase2 between the Golgi and the PM. We show here that V5-nSMase2 localizes at the PM and Golgi in MCF-7 cells and confirm relocalization of nSMase2 to the PM at confluence. Although cycloheximide (CHX) treatment partially inhibited the Golgi localization of GFP-nSMase2, recovery of GFP-nSMase2 to an intracellular compartment was still observed after photobleaching. Moreover, in the presence of CHX, GFP- and V5-nSMase2 co-localized with endosomal/recycling markers. In HEK293 cells, activation of either protein kinase C-alpha or betaII, with the phorbol ester PMA led to relocalization of both wild-type and inactive nSMase2 to the pericentrion, a PKC-dependent subset of recycling endosomes. Finally, inhibition of nSMase2 endocytosis by K + depletion reduced the intracellular pool of nSMase2 and increased nSMase2 activity resulting in elevated ceramide levels. Altogether, these results suggest that nSMase2 traffics from the Golgi to the PM as a membrane protein en route to the cell surface and recycles back to the Golgi through the endosomal/recycling compartment. Moreover, the recycling of nSMase2 from the PM is important for its catalytic regulation.     

Association of annexin 2 with recycling endosomes requires either calcium- or cholesterol-stabilized membrane domains

Annexin 2 is a Ca2+- and phospholipid-binding protein previously identified on endosomal membranes and the plasma membrane. Inferred from this location and its stimulatory effect on membrane transport annexin 2 has been proposed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the distribution of annexin 2 over the endosomal compartment and a characterization of the parameters governing this distribution. Towards this end we have devised an immunoisolation protocol to purify annexin 2-positive membrane vesicles from subcellular fractions of BHK cells containing early endosomes. We show that this approach leads to the isolation of intact endosomal vesicles containing internalized fluid-phase marker and that the immunoisolated membranes are positive for the transferrin receptor and Rab4 but not for the early endosomal antigen EEA1. A distinct and non-uniform distribution of annexin 2 over the early endosomal compartment is also observed in immunoelectron microscopy analyses of whole-mount specimens of BHK cells. Annexin 2 antibodies labeled transferrin receptor-containing tubular early endosomal structures, but not EEAl-positive endosomal vacuoles. We also observed that the Ca2+-independent association of annexin 2 with endosomal membranes was disrupted by the cholesterol-binding glycerid saponin, while Ca2+ could trigger annexin 2 binding to saponin-treated endosomal membranes. Thus, either Ca2+- or cholesterol-stabilized membrane domains are required for the binding of annexin 2 to endosomes suggesting that both factors may regulate this interaction.     

ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration.

A cDNA encoding a second zinc transporter (ZnT-2) was isolated from a rat kidney cDNA expression library by complementation of a zinc-sensitive BHK cell line. The protein predicted from the open reading frame of ZnT-2 cDNA has 359 amino acids and initiates with a CTG codon. It resembles ZnT-1 (a plasma membrane protein that stimulates zinc efflux) in overall topology in that it has six membrane-spanning domains, a histidine-rich intracellular loop and a long C-terminal tail; however, the overall amino acid identity is only 26%. Unlike ZnT-1, which is in the plasma membrane and lowers cellular zinc by stimulating zinc efflux, ZnT-2 is localized on vesicles and allows the zinc-sensitive BHK cells to accumulate zinc to levels that are much higher than non-transformed cells can tolerate. Zinc was visualized within these vesicles with zinquin, a zinc-specific fluorescent probe. The intracellular compartment that accumulates zinc is acidic as revealed by staining with acridine orange or LysoTracker. Prolonged exposure of cells expressing ZnT-2 to zinc causes an accretion of intracellular vesicles. We suggest that ZnT-2 protects these cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment.     

Workshop 7: 2

Glutamine, the preferred precursor for neurotransmitter glutamate, is likely to be the principal substrate for the neuronal System A transporter SAT1 in vivo. By measuring currents associated with SAT1 expression in Xenopus oocytes, we found that SAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine, alanine and the System A‐specific analogue 2‐(methylamino) isobutyrate, each with K_0.5 of 0.3–0.5 mm . Amino acid transport is driven by the Na⁺ electrochemical gradient. Kinetic data indicates that Na⁺/cotransport comprises the ordered binding first of Na⁺ (a voltage‐dependent step), then alanine, then simultaneous translocation. Li⁺ (but not H⁺) can substitute for Na⁺ but results in reduced V_max. In the absence of amino acid, SAT1 mediates a cation leak with selectivity Na⁺, Li⁺, H⁺, K⁺. The temperature‐dependence of the leak current (E_a = 17 ± 3 kcal/mol) is consistent with carrier‐mediated Na⁺ uniport activity (cf 13 ± 2 kcal/mol for Na⁺/alanine cotransport) but the leak does not saturate at physiological [Na⁺], suggesting channel activity. Despite a Na⁺ Hill coefficient of 1, we obtained Na⁺/amino acid coupling coefficients greater than 1 from simultaneous measurement of charge and [³H]alanine or [³H]glutamine uptake. Interpretation of these data is model‐dependent and consistent with either (1) an all‐carrier model in which Na⁺/amino acid cotransport is thermodynamically coupled 2 : 1, cotransport is preferred over Na⁺ uniport, and in which there is little cooperativity between Na⁺ binding events, or (2) 1 : 1 coupling in parallel with an always‐on Na⁺ channel activity. In either scenario, the presence of SAT1 at the plasma membrane and resultant Na⁺ fluxes will place a significant energy burden on the cell.     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

Activation of protein kinase C zeta induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle

Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.