Biomedical subjects

A Klip

Publications and source records attributed to A Klip.

At least 55 records · Page 3Linked to original sources

PubMed1998

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.

Actin Cytoskeleton↗

PubMed1998

Phosphatidylinositol 4-kinase, but not phosphatidylinositol 3-kinase, is present in GLUT4-containing vesicles isolated from rat skeletal muscle.

Insulin stimulates the rate of glucose transport into muscle and adipose cells by translocation of glucose transporter (GLUT4)-containing vesicles from an intracellular storage pool to the surface membrane. This event is mediated through the insulin receptor substrates (IRSs), which in turn activate phosphatidylinositol (PI) 3-kinase isoforms. It has been suggested that insulin causes attachment of PI 3-kinases to the intracellular GLUT4-containing vesicles in rat adipose cells. Furthermore, it has also been shown that GLUT4-containing vesicles in adipose cells contain a PI 4-kinase. In the present study we investigate whether GLUT4-containing vesicles isolated from rat skeletal muscle display PI 3-kinase and/or PI 4-kinase activities. Insulin stimulation caused a rapid increase (5-15-fold increase compared with control) in the intracellular cytosolic IRS-1-associated PI-3 kinase activity. This PI 3-kinase activity was also present in a membrane preparation containing the insulin-regulatable pool of GLUT4 transporters. However, when GLUT4-containing vesicles were isolated by immunoprecipitation from basal and insulin-stimulated (3 min) skeletal muscle, the vesicles displayed PI 4-kinase, but not PI 3-kinase, activity. Insulin did not regulate the PI 4-kinase activity in the GLUT4-containing vesicles. In conclusion, GLUT4-containing vesicles from rat skeletal muscle contain a PI 4-kinase, but not a PI 3-kinase. It is suggested that, in skeletal muscle, insulin causes activation of the IRS/PI 3-kinase complex in an intracellular membrane compartment associated closely with the GLUT4-containing vesicles, but not in the GLUT4-containing vesicles themselves.

1-Phosphatidylinositol 4-Kinase↗

PubMed1998

Binary interactions of the SNARE proteins syntaxin-4, SNAP23, and VAMP-2 and their regulation by phosphorylation.

The SNARE hypothesis proposes that synaptic vesicles dock at presynaptic membranes via interactions among the vesicular, integral membrane proteins VAMP (vesicle-associated membrane protein) and synaptotagmin and the target membrane proteins SNAP25 (synaptosome-associated protein with an Mr of 25 kDa) and syntaxin-1. Non-neuronal cells express isoforms of these proteins, believed to mediate secretory vesicle docking and/or fusion. Secretion in neuronal and non-neuronal systems differs in time course, Ca2+ dependence, and regulatory input. It is not known whether the non-neuronal protein isoforms form complexes akin to those of their neuronal counterparts. In this study, we defined the binding characteristics of three SNARE proteins: SNAP23, VAMP-2, and syntaxin-4. Binary, saturable interactions among all three partners (VAMP-2-syntaxin-4, VAMP-2-SNAP23, and SNAP23-syntaxin-4) were measured in vitro. Unlike its neuronal counterpart, SNAP23 did not potentiate VAMP-2 binding to its putative t-SNARE partner, syntaxin-4. The susceptibility of SNARE proteins to phosphorylation by exogenous kinases and their impact on binary interactions were explored. Syntaxin-4 was efficiently phosphorylated by casein kinase II (CKII) and cAMP-dependent protein kinase (PKA) (incorporating 0.8 and 3.9 mol of phosphate/mol of syntaxin-4, respectively), while syntaxin-1 was only strongly phosphorylated by CKII. Each of the syntaxin isoforms was weakly phosphorylated by protein kinase C (PKC) (<0.05 mol of phosphate/mol of syntaxin-4). Importantly, PKA but not casein kinase II phosphorylation of syntaxin-4 disrupted its binding to SNAP23. We hypothesize that PKA may modulate syntaxin-4-dependent SNARE complex formation to regulate exocytosis in non-neuronal cells.

Animals↗

PubMed1998

Unique mechanism of GLUT3 glucose transporter regulation by prolonged energy demand: increased protein half-life.

L6 muscle cells survive long-term (18 h) disruption of oxidative phosphorylation by the mitochondrial uncoupler 2,4-dinitrophenol (DNP) because, in response to this metabolic stress, they increase their rate of glucose transport. This response is associated with an elevation of the protein content of glucose transporter isoforms GLUT3 and GLUT1, but not GLUT4. Previously we have reported that the rise in GLUT1 expression is likely to be a result of de novo biosynthesis of the transporter, since the uncoupler increases GLUT1 mRNA levels. Unlike GLUT1, very little is known about how interfering with mitochondrial ATP production regulates GLUT3 protein expression. Here we examine the mechanisms employed by DNP to increase GLUT3 protein content and glucose uptake in L6 muscle cells. We report that, in contrast with GLUT1, continuous exposure to DNP had no effect on GLUT3 mRNA levels. DNP-stimulated glucose transport was unaffected by the protein-synthesis inhibitor cycloheximide. The increase in GLUT3 protein mediated by DNP was also insensitive to cycloheximide, paralleling the response of glucose uptake, whereas the rise in GLUT1 protein levels was blocked by the inhibitor. The GLUT3 glucose transporter may therefore provide the majority of the glucose transport stimulation by DNP, despite elevated levels of GLUT1 protein. The half-lives of GLUT3 and GLUT1 proteins in L6 myotubes were determined to be about 15 h and 6 h respectively. DNP prolonged the half-life of both proteins. After 24 h of DNP treatment, 88% of GLUT3 protein and 57% of GLUT1 protein had not turned over, compared with 25% in untreated cells. We conclude that the long-term stimulation of glucose transport by DNP arises from an elevation of GLUT3 protein content associated with an increase in GLUT3 protein half-life. These findings suggest that disruption of the oxidative chain of L6 muscle cells leads to an adaptive response of glucose transport that is distinct from the insulin response, involving specific glucose transporter isoforms that are regulated by different mechanisms.

2,4-Dinitrophenol↗

PubMed1998

Identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (VAP-33): a broadly expressed protein that binds to VAMP.

We report the identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein (hVAP-33) that has been implicated in neuronal exocytosis in Aplysia californica. This hVAP-33 shared 50% amino acid identity with the A. californica form and had similar length, structural organization and VAMP-binding abilities. However, in contrast with the neuron-specific expression seen in A. californica, hVAP-33 was broadly expressed, suggesting possible roles in vesicle fusion in both neuronal and non-neuronal cells.

Amino Acid Sequence↗

PubMed1998

GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay.

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 microM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs.

Androstadienes↗

PubMed1998

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.

3T3 Cells↗

PubMed1998

Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate.

Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4, 5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor's action.

3T3 Cells↗

PubMed1998

Perturbation of dynamin II with an amphiphysin SH3 domain increases GLUT4 glucose transporters at the plasma membrane in 3T3-L1 adipocytes. Dynamin II participates in GLUT4 endocytosis.

The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. Glutathione S-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake.

3T3 Cells↗

PubMed1998

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.

Androstadienes↗

PubMed1998

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

Androstadienes↗

PubMed1998

Molecular mechanisms involved in GLUT4 translocation in muscle during insulin and contraction stimulation.

Studies in mammalian cells have established the existence of numerous intracellular signaling cascades that are critical intermediates in the regulation of various biological functions. Over the past few years considerable research has shown that many of these signaling proteins are expressed in skeletal muscle. However, the detailed mechanisms involved in the regulation of glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface membrane in response to insulin and contractions in skeletal muscle are not well understood. In the present essay we report three different approaches to unravel the GLUT4 translocation mechanism: 1. specific pertubation of the insulin and/or contraction signaling pathways; 2. characterization of the protein composition of GLUT4-containing vesicles with the expectation that knowledge of the constituent proteins of the vesicles may help in understanding their trafficking; 3. degree of co-immunolocalization of the GLUT4 glucose transporters with other membrane marker proteins assessed by immunofluorescense and electron microscopy.

Animals↗

PubMed1998

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.

3T3 Cells↗

PubMed1998

Opposite effects of insulin on focal adhesion proteins in 3T3-L1 adipocytes and in cells overexpressing the insulin receptor.

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.

3T3 Cells↗

PubMed1998

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.

2,4-Dinitrophenol↗

PubMed1998

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.

Androstadienes↗

PubMed1998

Regulation of the Na+/K+-ATPase by insulin: why and how?

The sodium-potassium ATPase (Na+/K+-ATPase or Na+/K+-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na+ from cells in exchange for K+ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits (alpha and beta) each expressed in several isoforms. Many hormones regulate Na+/K+-ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na+/K+-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na+ concentration, altered Na+ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na+/K+-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na+/K+-ATPase control by insulin in diabetes and related disorders is addressed.

Animals↗

PubMed1997

SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion.

The synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in neurons and endocrine cells. It has been shown to play an important role in the release mechanism of neurotransmitters and peptide hormones, including insulin. Thus, when insulin-secreting cells are permeabilized and treated with botulinum neurotoxin E (BoNT/E), SNAP-25 is hydrolyzed, and insulin secretion is inhibited. Recently SNAP-23, a more generally expressed isoform of SNAP-25, has been described. The functional role of SNAP-23 has not been investigated to date. It is now shown that SNAP-23 is resistant to cleavage by BoNT/E. It was therefore possible to test whether transfection of HIT (transformed pancreatic B-) cells with SNAP-23 reconstitutes insulin release from BoNT/E treated cells, in which SNAP-25 is inactivated by the toxin. The results show that SNAP-23 is able to replace SNAP-25 when it is overexpressed. While these results demonstrate that SNAP-23 is a functional homologue of SNAP-25, able to function in regulated exocytosis, they indicate that SNAP-23 may be inefficient in this process. This suggests that both isoforms may have their own specific binding partners and discrete, albeit mechanistically similar, functional roles within the cell.

Animals↗