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P F Pilch

Publications and source records attributed to P F Pilch.

At least 37 records · Page 2Linked to original sources
PubMed1997

Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of aggregated beta-amyloid peptide, presumed to play a pathogenic role, and by preferential loss of neurons that express the 75-kD neurotrophin receptor (p75NTR). Using rat cortical neurons and NIH-3T3 cell line engineered to stably express p75NTR, we find that the beta-amyloid peptide specifically binds the p75NTR. Furthermore, 3T3 cells expressing p75NTR, but not wild-type control cells lacking the receptor, undergo apoptosis in the presence of aggregated beta-amyloid. Normal neural crest-derived melanocytes that express physiologic levels of p75NTR undergo apoptosis in the presence of aggregated beta-amyloid, but not in the presence of control peptide synthesized in reverse. These data imply that neuronal death in Alzheimer's disease is mediated, at least in part, by the interaction of beta-amyloid with p75NTR, and suggest new targets for therapeutic intervention.

3T3 Cells↗
PubMed1997

Sortilin is a major protein component of Glut4-containing vesicles.

In fat and skeletal muscle cells, glucose transporter isoform 4 (Glut4) is translocated to the cell surface in response to insulin via a system of specialized recycling vesicles. Besides Glut4, these vesicles include the novel insulin-regulatable aminopeptidase, receptors for insulin-like growth factor-II/Man-6-phosphate and transferrin, and a glycoprotein with the molecular mass of 110 kDa. We report here by the criteria of the partial protein sequencing and subsequent cDNA cloning that glycoprotein 110, the last unidentified major protein component of Glut4-containing vesicles, is sortilin, a novel type I receptor-like protein recently cloned from human brain (Petersen, C. M., Nielsen, M. S., Nykjar, A., Jacobsen, L., Tommerup, N., Rasmussen, H. H., Roigaard, H., Gliemann, J., Madsen, P., and Moestrup, S. K. (1997) J. Biol. Chem. 272, 3599-3605). This protein is highly expressed in fat, brain, and lung and is dramatically up-regulated during differentiation of adipocytes in vitro.

3T3 Cells↗
PubMed1997

Conformational changes of the insulin receptor upon insulin binding and activation as monitored by fluorescence spectroscopy.

We have characterized the changes in intrinsic fluorescence that the insulin receptor undergoes upon ligand binding and autophosphorylation. The binding of insulin to its receptor results in an increase in the receptor's fluorescence intensity, emission energy and anisotropy. We monitored the time course of the anisotropy change, and these data, coupled with studies monitoring the energy transfer from insulin receptor tryptophan donors to a fluorescent-labeled insulin, allowed us to conclude that the change in anisotropy is due to a conformational change in the receptor induced by hormone binding. Since insulin association is very fast, the time course also allowed us to estimate the slower rate of formation of this conformationally-altered state. The time course of receptor autophosphorylation was measured under similar conditions and was found to be similar to the ligand-induced anisotropy time course. The simultaneous use of two fluorescent-labeled insulin analogs also allowed us to assess the maximum distance between the two hormones bound to the receptor. Addition of ATP produces a large, seemingly instantaneous increase in anisotropy. Our observation that ATP binds to the insulin receptor in the presence and absence of insulin supports the idea that the conformational change produced by insulin binding increases the rate of autophosphorylation rather than increases ATP affinity. A suggested model for these changes is presented.

3T3 Cells↗
PubMed1997

Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction.

A number of studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is associated with profound insulin resistance in adipocytes and may also play a critical role in the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. Reports on the mechanism of TNF-alpha action have been somewhat contradictory. GLUT4 down-regulation has been implicated as a possible cause of insulin resistance as has been the reduced kinase function of the insulin receptor. Here we examine the effects of tumor necrosis factor on the protein components thought to be involved in insulin-stimulated glucose transport in adipocytes, namely the insulin receptor, its major substrate IRS-1, and the insulin responsive glucose transporter GLUT4. Prolonged exposure (72-96 h) of 3T3-L1 adipocytes to TNF-alpha causes a substantial reduction (>80%) in IRS-1 and GLUT4 mRNA and protein as well as a lesser reduction (>50%) in the amount of the insulin receptor. Nevertheless, the remaining proteins appear to be biochemically indistinguishable from those in untreated adipocytes. Both the insulin receptor and IRS-1 are tyrosine-phosphorylated to the same extent in response to acute insulin stimulation following cellular TNF-alpha exposure. Furthermore, the ability of the insulin receptor to phosphorylate exogenous substrate in the test tube is also normal following its isolation from TNF-alpha-treated cells. These results are confirmed by the reduced but obvious level of insulin-dependent glucose transport and GLUT4 translocation observed in TNF-alpha-treated adipocytes. We conclude that the insulin resistance of glucose transport in 3T3-L1 adipocytes exposed to TNF-alpha for 72-96 h results from a reduced amount in requisite proteins involved in insulin action. These results are consistent with earlier studies indicating that TNF-alpha reduces the transcriptional activity of the GLUT4 gene in murine adipocytes, and reduced mRNA transcription of a number of relevant genes may be the general mechanism by which TNF-alpha causes insulin resistance in adipocytes.

3T3 Cells↗
PubMed1996

The insulin-like growth factor II/mannose 6-phosphate receptor utilizes the same membrane compartments as GLUT4 for insulin-dependent trafficking to and from the rat adipocyte cell surface.

The insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptor recycles in adipose cells between the cell surface and an intracellular storage pool, and the rate of this trafficking is markedly enhanced by insulin. We show here that the IGF-II/Man-6-P receptor is a constituent of the GLUT4-containing compartment ("GLUT4 vesicles") where it represents gp230, a major recycling protein detected earlier by cell surface biotinylation (Kandror, K. V., and Pilch, P. F. (1994) J. Biol. Chem. 269, 138-142). The GLUT4 vesicles include 10-15% of the total and all of the acutely insulin-responsive recycling population of the IGF-II/Man-6-P receptor. The main part of the IGF-II/Man-6-P receptor population is excluded from the pathway of GLUT4 trafficking and either resides permanently in intracellular membranes or has a much slower rate of cycling to the cell surface. Thus, GLUT4 vesicles mediate the insulin-dependent delivery to the cell surface of the IGF-II/Man-6-P receptor as well as the other recyclable proteins with extracellular functional domains (GLUT4 and the aminopeptidase gp160).

Adipose Tissue↗
PubMed1996

The expression and regulation of STATs during 3T3-L1 adipocyte differentiation.

STATs (Signal Transducers and Activators of Transcription) comprise a family of transcription factors that reside in the cytoplasm of resting cells. In response to a variety of stimuli, STATs become tyrosine-phosphorylated and translocate to the nucleus where they mediate transcriptional regulation. We have used the 3T3-L1 murine cell line to examine the expression of STAT proteins as a function of their differentiation into adipocytes. The expression of STATs 1, 3, and 5, but not of STAT 6, is markedly elevated in adipocytes as compared with their fibroblast precursors. Exposure of 3T3-L1 preadipocytes to tumor necrosis factor alpha (TNF alpha) blocks their differentiation into adipocytes. Therefore, we examined STAT expression as a function of differentiation in the presence of this cytokine. The expression of STATs 1 and 5 is markedly attenuated in the presence of TNF alpha, whereas STAT 3 expression is unaffected by this treatment. Only STAT 1 is down-regulated by TNF alpha in fully differentiated cells. Thus, although the expression of STATs 1, 3, and 5 is markedly enhanced upon differentiation, only STAT 5 expression is tightly correlated with the adipocyte phenotype. These data suggest that STAT 5, and possibly STAT 1, could be potential inducers of tissue-specific genes, which contribute to the development and maintenance of the adipocyte phenotype.

3T3 Cells↗
PubMed1996

Glut4 is targeted to specific vesicles in adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue.

Adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue (Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4-containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulin-sensitive vesicles as in normal cells.

Adipocytes↗
PubMed1996

Stimulation of C2C12 myoblast growth by basic fibroblast growth factor and insulin-like growth factor 1 can occur via mitogen-activated protein kinase-dependent and -independent pathways.

It is now well-recognized that the mitogen-activated protein (MAP) kinase cascade facilitates signaling from an activated tyrosine kinase receptor to the nucleus. In fact, an increasing number of extracellular effectors have been reported to activate the MAP kinase cascade, with a significant number of cellular responses attributed to this activation. We set out to explore how two extracellular effectors, basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-1), which have both been reported to activate MAP kinase, generate quite distinct cellular responses in C2C12 myoblasts. We demonstrate here that bFGF, which is both a potent mitogen and inhibitor of myogenic differentiation, is a strong MAP kinase agonist. By contrast, IGF-1, which is equally mitogenic for C2C12 cells but ultimately enhances the differentiated phenotype, is a weak activator of the MAP kinase cascade. We further demonstrate that IGF-1 is a potent activator of both insulin receptor substrate IRS-1 tyrosyl phosphorylation and association of IRS-1 with activated phosphatidylinositol 3-kinase (PI 3-kinase). Finally, use of the specific MAP kinase kinase inhibitor, PD098059, and wortmannin, a PI 3-kinase inhibitor, suggests the existence of an IGF-1-induced, MAP kinase-independent signaling event which contributes to the mitogenic response of this factor, whereas bFGF-induced mitogenesis appears to strongly correlate with activation of the MAP kinase cascade.

Androstadienes↗
PubMed1996

Compartmentalization of protein traffic in insulin-sensitive cells.

Insulin-sensitive cells, adipocytes and myocytes, translocate a number of intracellular proteins to the cell surface in response to insulin. Among these proteins are glucose transporters 1 and 4 (GLUT-1 and GLUT-4, respectively), receptors for insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) and transferrin, the aminopeptidase gp 160, caveolin, and a few others. In the case of insulin-activated glucose transport, this translocation has been proven to be the major, if not the only regulatory mechanism of this process. It seems likely that the cell surface recruitment of the IGF-II/Man-6-P and transferrin receptors also serves the nutritional needs of cells, whereas the physiological role of the aminopeptidase gp160 remains uncertain. Analysis of the compartmentalization and trafficking pathways of translocatable proteins in fat cells identified more than one population of recycling vesicles, although all have identical sedimentation coefficients and buoyant densities in vitro. GLUT-4-containing vesicles include essentially all the intracellular GLUT-4, gp160, and the acutely recycling populations of receptors for IGF-II/Man-6-P and transferrin. Besides these proteins, which can be considered as vesicle "cargo", GLUT-4-containing vesicles have other components, like secretory carrier-associated membrane proteins (SCAMP), Rab(s), and vesicle-associated membrane protein (VAMP)/cellubrevin, which are ubiquitous to secretory vesicles and granules from different tissues. GLUT-1 and caveolin are excluded from GLUT-4-containing vesicles and form different vesicular populations of unknown polypeptide composition. In skeletal muscle, two independent populations of GLUT-4-containing vesicles are found, insulin sensitive and exercise sensitive, which explains the additive effect of insulin and exercise on glucose uptake. Both vesicular populations are similar to each other and to analogous vesicles in fat cells.

Adipocytes↗
PubMed1996

In vivo effects of dexamethasone and sucrose on glucose transport (GLUT-4) protein tissue distribution.

Tissue-specific changes in GLUT-4 were compared in the following three different rat models by inducing varying degrees of hyperinsulinemia with or without hyperglycemia and hypertriglyceridemia: 1) sucrose feeding (Suc), 2) subcutaneous dexamethasone administration (Dex), and 3) a combination of both treatments (Dex/Suc). Suc raised circulatory insulin and triglyceride levels without affecting plasma glucose, whereas both Dex and Dex/Suc induced significant hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. In adipocytes and skeletal muscle, Suc feeding was not associated with any change in total cellular GLUT-4 levels. However, Suc induced a sevenfold increase in fat cell plasma membrane GLUT-4 levels in the basal state and inhibited GLUT-4 translocation in response to insulin. Administration of Dex or Dex/Suc diminished GLUT-4 expression in fat cells, increased it in skeletal muscle, but did not induce any change in heart. Similar to Suc feeding, Dex and Dex/Suc also increased the amount of GLUT-4 detected at the plasma membrane of adipocytes in the basal state and inhibited GLUT-4 translocation in response to insulin. These results emphasize the specific regulation of GLUT-4 in insulin-sensitive tissues.

Adipose Tissue↗
PubMed1995

Intermolecular phosphorylation between insulin holoreceptors does not stimulate substrate kinase activity.

We photocoupled benzoylphenylalanineB25, B29 epsilon-biotin insulin (BBpa-insulin) to native insulin receptors to obtain a uniform receptor population with covalently bound, non-dissociable ligand. We employed BBpa-insulin-bound and autophosphorylated (activated) receptor to phosphorylate substrate insulin receptor under conditions where the substrate receptor never interacts with insulin. The substrate receptor becomes phosphorylated in this inter-receptor fashion and reaches a phosphorylation state 50% of the maximal obtainable by autophosphorylation. However, this phosphorylation does not activate the substrate receptor to any measurable degree. We conclude that intermolecular phosphorylation of the insulin holoreceptors is unlikely to be of physiological significance.

3T3 Cells↗
PubMed1995

The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle.

(1) In this study we have determined the distribution of various membrane proteins involved in insulin-activated glucose transport in T-tubules and in sarcolemma from rat skeletal muscle. Two independent experimental approaches were used to determine the presence of membrane proteins in T-tubules: (i) the purification of T-tubules free from sarcolemmal membranes by lectin agglutination, and (ii) T-tubule vesicle immunoadsorption. These methods confirmed that T-tubules from rat skeletal muscle were enriched with dihydropyridine receptors and tt28 protein and did not contain the sarcolemmal markers dystrophin or beta 1-integrin. Both types of experiments revealed an abundant content of GLUT4 glucose carriers, insulin receptors and SCAMPs (secretory carrier membrane proteins) in T-tubule membranes. (2) Acute administration in vivo of insulin caused an increased abundance of GLUT4 in T-tubules and sarcolemma. On the contrary, insulin led to a 50% reduction in insulin receptors present in T-tubules and in sarcolemma, demonstrating that insulin-induced insulin receptor internalization affects T-tubules in the muscle fibre. The alteration in the content of GLUT4 and insulin receptors in T-tubules was a consequence of insulin-induced redistribution of these proteins. SCAMPs also redistributed in muscle membranes in response to insulin. They were recruited by insulin from intracellular high-density fractions to intracellular lighter-density fractions and to the cell surface, showing a pattern of insulin-induced cellular redistribution distinct from those of GLUT4 and the insulin receptor. (3) In conclusion, the T-tubule is a cell-surface target for membrane proteins involved in recycling such as SCAMPs or for membrane proteins that acutely redistribute in response to insulin such as GLUT4 or insulin receptors.

Animals↗
PubMed1995

Identification and characterization of an exercise-sensitive pool of glucose transporters in skeletal muscle.

Augmentation of glucose transport into skeletal muscle by GLUT4 translocation to the plasma and T-tubule membranes can be mediated independently by insulin and by contraction/exercise. Available data suggest that separable pools of intracellular GLUT4 respond to these two stimuli. To identify and characterize these pools, we fractionated skeletal muscle membranes in a discontinuous sucrose density gradient. Fractions of 32 and 36% sucrose exhibited the highest enrichment of GLUT4 and were independently responsive to insulin and exercise, respectively. The combination of the two stimuli depleted both GLUT4 fractions simultaneously. Both vesicle populations contained the gp160 aminopeptidase, whose expression had previously been shown to be specific to muscle and fat and restricted to GLUT4 vesicles in the latter tissue. In muscle, gp160 translocates exactly as does GLUT4 in response to insulin and exercise. The contraction- and insulin-sensitive GLUT4 pools also contained secretory component-associated membrane protein/glucose transporter vesicle triplet but not GLUT1 and caveolin. Immunoadsorption of the two pools followed by silver staining did not reveal any obvious difference in their major protein components. On the other hand, sedimentational analysis in sucrose velocity gradients revealed that the insulin-sensitive GLUT4 vesicles had a larger sedimentation coefficient than the exercise-sensitive vesicles. Thus, the separation of the two intracellular GLUT4 pools should be useful in dissecting what are likely to be different signal transduction pathways that mediate their translocation to the cell surface.

Aminopeptidases↗
PubMed1995

Comparison of glucose-transporter-containing vesicles from rat fat and muscle tissues: evidence for a unique endosomal compartment.

Insulin-sensitive tissues (fat and muscle) express a specific isoform of glucose-transporter protein, GLUT4, which normally resides in intracellular vesicular structures and is translocated to the cell surface in response to insulin. Here we provide a biochemical comparison of GLUT4-containing structures from fat and muscle cells. We demonstrate that, in spite of totally different protocols for cell homogenization and fractionation used for adipocytes as compared with skeletal-muscle tissue, GLUT4-containing vesicles from both sources have identical buoyant densities, sedimentation coefficients, and a very similar, if not identical, protein composition. The individual proteins first identified in GLUT4-containing vesicles from adipocytes (GTV3/SCAMPs proteins and aminopeptidase gp160) are also present in the analogous vesicles from muscle. Intracellular microsomes from rat adipocytes also contain GLUT1, a ubiquitously expressed glucose-transporter isoform. GLUT1 has not been detected in intracellular vesicular pool(s) from skeletal-muscle cells, probably because of its low abundance there. GLUT1 in adipocytes is excluded from GLUT4-containing vesicles, but is found in membrane structures which are indistinguishable from the former by all methods tested and demonstrate the same type of regulation by insulin. That is, the GLUT1- and GLUT4-containing vesicles have identical densities and sedimentation coefficients in sucrose gradients, and translocate to the cell surface in response to hormonal exposure. Also, we describe a simple procedure for the purification of native glucose-transporter vesicles from rat adipocytes. Overall, our data suggest the existence of a unique endosomal compartment in fat and muscle cells which is functionally and compositionally different from other microsomal vesicles and which is responsible for insulin-sensitive glucose transport in these tissues.

Adipose Tissue↗
PubMed1995

Dynamics of signaling during insulin-stimulated endocytosis of its receptor in adipocytes.

Insulin causes rapid insulin receptor autophosphorylation, receptor endocytosis, and phosphorylation of its principle substrate (IRS-1). Using rat adipocytes, we studied the dynamics of receptor autophosphorylation, the kinase activity, and the IRS-1 phosphorylation state relative to the subcellular localization of these proteins. After 2 min of insulin exposure, the specific phosphotyrosine content of the insulin receptor in the internal membranes (IM) peaks at a level 5-6-fold higher than the plasma membrane (PM) receptor and then declines after 5-8 min to a level similar to the PM receptor. The exogenous kinase activity of these receptors exactly mirrored their phosphotyrosine content. The distribution of IRS-1 is 80% cytosolic, 20% IM-associated, and essentially undetectable in the PM. The phosphorylation state of IRS-1 in the IM parallels that of the insulin receptor, but cytosolic IRS-1 phosphorylation remains constant. Insulin-dependent GLUT4 translocation to the PM occurs after the peak of IRS-1 phosphorylation. The data are consistent with the hypothesis that insulin action may be mediated by receptor internalization and interaction with its substrate(s) associated with internal membranes. A small fraction of phosphorylated insulin receptors is sufficient for signal transduction. The dephosphorylation of the insulin receptor and IRS-1 in the IM appears to be a concerted process, possibly mediated by the same enzyme.

Adipocytes↗
PubMed1995

Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.

3T3 Cells↗
PubMed1994

The major protein of GLUT4-containing vesicles, gp160, has aminopeptidase activity.

A glycoprotein of M(r) 160,000 (gp160) represents the major protein component of GLUT4-containing vesicles other than GLUT4 itself. These vesicles are the tissue-specific secretory-like microsomal structures that are responsible for insulin-dependent translocation of GLUT4 to the cell surface in fat and muscle cells. Our previous studies demonstrated that the expression of gp160 is also limited to fat and muscle tissues, where it is localized exclusively in GLUT4-containing vesicles, and thus, it represents a marker protein for insulin-activated glucose transport. We demonstrate here that gp160 has structural homology to aminopeptidase N and possesses aminopeptidase activity in vitro. Since aminopeptidase activity has also been found in a variety of secretory type vesicles from other tissues, it may be essential for the organization and functioning of secretory machinery in different cell types.

Adipocytes↗