Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1104685 on April 21, 2005

Published online before print April 21, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1104685v1
78/1/187    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Misra, U. K.
Right arrow Articles by Pizzo, S. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Misra, U. K.
Right arrow Articles by Pizzo, S. V.
(Journal of Leukocyte Biology. 2005;78:187-194.)
© 2005 by Society for Leukocyte Biology

Up-regulation of GRP78 and antiapoptotic signaling in murine peritoneal macrophages exposed to insulin

Uma Kant Misra and Salvatore Vincent Pizzo1

Department of Pathology, Duke University Medical Center, Durham, North Carolina

1 Correspondence: Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710. E-mail: Pizzo001{at}mc.duke.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The unfolded protein response pathway (UPR) compensates for excessive protein accumulation in the endoplasmic reticulum (ER). As insulin induces global protein synthesis, it may cause accumulation of unfolded proteins in the ER, thus triggering UPR. We assessed UPR activation in insulin-treated murine peritoneal macrophages using a number of markers including 78 kDa glucose response protein (GRP78), X-box-binding protein (XBP)-1, pancreatic ER kinase (PERK), eukaryotic initiation factor 2 (eIF2){alpha}, and growth arrest and DNA damage (GADD)34. Exposure of cells to insulin activated UPR, as evidenced by an increased expression of GRP78, XBP-1, phosphorylated PERK (p-PERK), and p-eIF2{alpha}. The insulin-induced, elevated expression of GRP78 was comparable with that observed with tunicamycin, a classical inducer of ER stress. Concomitantly, insulin also up-regulated prosurvival mechanisms by elevating GADD34 and elements of the antiapoptotic pathway including Bcl-2, X-linked inhibitor of apoptosis, and phosphorylated forkhead transcription factor. In conclusion, we show here that insulin treatment does cause ER stress in macrophages, but insulin-dependent mechanisms overcome this ER stress by up-regulating UPR and the antiapoptotic pathway to promote cell survival.

Key Words: unfolded protein response • apoptosis • Akt • FKHR • CREB • regulation of protein synthesis • endoplasmic reticulum • regulation of protein catabolism


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pleotropic effects of insulin are initiated upon its binding to its receptor, which is ubiquitously expressed in mammalian cells. Insulin receptor-binding activates the tyrosine kinase activity of its ß-subunit, which then phosphorylates a number of intracellular substrates initiating activation of the phosphoinositide-3 kinase (PI-3K) and Ras-mitogen-activated protein kinase (MAPK) signaling pathways [1 2 3 4 ]. Activation of PI-3K is necessary for many of the effects of insulin, including the protein kinase cascade involving Akt. Ras can also activate PI-3K through direct interaction with its p110 catalytic subunit. PI-3K functions in many cellular responses, including the regulation of transcription, mitogenesis, and programmed cell death [4 ]. Insulin is an anabolic hormone, which greatly enhances global protein synthesis. This insulin-induced, accelerated protein synthesis requires a balance between polypeptide synthesis and proper protein folding in the endoplasmic reticulum (ER). Any compromise in this balance would result in the accumulation of unfolded proteins in the ER causing ER "stress". Mammalian cells protect themselves from such ER stress by triggering an unfolded protein response (UPR), which coordinates a broad down-regulation of protein synthesis with increased expression of various gene products including ER resident molecular chaperones that promote protein folding and degradation of unfolded proteins (see refs. [5 6 7 8 9 10 11 12 ] and references therein). UPR can also lead to the arrest of cell growth, and chronic ER stress leads to cell death (refs. [5 6 7 8 9 10 11 12 ] and references therein).

Physiological conditions that induce UPR caused by protein misfolding include the differentiation and development of professional secretory cells, altered metabolic conditions, mutations in the genes encoding secretory or transmembrane proteins, and infection by certain pathogens (see ref. [5 ] and references therein). UPR can also be induced experimentally, such as by inhibition of NH2-linked glycosylation in the ER, depletion of ER calcium stores, and reductive stress [5 6 7 8 9 10 11 12 ]. Three ER resident transmembrane proteins have been identified as the proximal sensors of the presence of ER stress. These are pancreatic ER kinase (PERK), endonuclease inositol requiring-1 (IRE1{alpha} and -ß), and the basic leucine zipper transcription factor activating transcription factor 6 (ATF6{alpha} and -ß) [5 6 7 8 9 10 11 12 ]. Under ER stress, IRE1 and PERK undergo homodimerization, autophosphorylation, and activation. Accumulation of unfolded proteins in the ER lumen leads to the transit of ATF6 to the Golgi complex, where it is cleaved into an active transcription factor [13 ]. The effect of the activation of these molecules is twofold. There is an up-regulation of the genes encoding ER resident chaperones and proteins involved in ER-associated protein degradation. Concomitantly, there is a down-regulation of protein synthesis and reduced influx of nascent proteins into the ER [5 6 7 8 9 10 11 12 13 ]. UPR is an adaptive response, which progresses through a series of strictly controlled steps to alleviate the problem of unfolded proteins. The activation of all three protein components of UPR depends on their dissociation from 78 kDa glucose response protein (GRP78) [5 ], which is a calcium-binding chaperone protein with cell-protective properties [13 14 15 ]. Overexpression and antisense approaches in cell systems show that GRP78 can protect against cell death caused by disturbance of ER homeostasis. Cellular stress results in an induction of not only GRP78 but also other coordinately regulated genes [5 , 13 ]. Thus, GRP78 has been used as a biomarker of ER stress. In this study, we addressed the question of whether exposure of peritoneal macrophages causes ER stress as a result of insulin-induced, accelerated global protein synthesis and whether activation of UPR occurs to protect these cells from such adverse effects. We evaluated insulin-induced, physiological ER stress and UPR by studies of GRP78, various components of UPR, and insulin-induced antiapoptotic pathway activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The sources of cell culture and Western blotting materials have been described previously [16 17 18 ]. GRP78 protein and antibodies against GRP78 were procured from Stressgen Bioreagents, (Victoria, BC, Canada). Antibodies against X-box-binding protein (XBP)-1, growth arrest and DNA damage (GADD)34, GADD153, and 14-3-3 were obtained from Santa Cruz Biotechnology (CA). Antibodies against phosphorylated extracellular signal-regulated kinase (p-ERK)1/2, p38 MAPK, Jun N-terminal kinase (JNK), p-p85 Src homology 2-binding protein of PI-3K, phosphoinositide-dependent protein kinase-1 (PDK1), Akt at Thr308 or Ser473, p70 s6k, cyclic AMP (cAMP) response element-binding protein (CREB) at Ser133, PERK phosphorylated at Thr980, eukaryotic initiation factor 2 (eIF2){alpha} phosphorylated at Ser51 of the {alpha} chain, eIF2{alpha}, Bcl-2 phosphorylated at Ser70, X-linked inhibitor of apoptosis (XIAP) phosphorylated glycogen synthase kinase 3 (GSK3){alpha}/ß at Ser219 phosphorylated forkhead transcription factor (FKHR) at Ser256, and FKHR protein were obtained from Cell Signaling Technology (Beverly, MA). Insulin and tunicamycin were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture
Thioglycollate-elicited peritoneal macrophages were obtained from pathogen-free, 6-week-old c57BL/6 mice (National Cancer Institute, Frederick, MD) in Hanks’ balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.4, and 3.5 mM NaHCO3 (HHBSS) at 4°C. The cell suspension was centrifuged at 800 g for 5 min at 4°C. The pellet was washed once with chilled HHBSS and suspended in a volume of RPMI-1640 medium containing 2 mM glutamine, penicillin (12.5 units/ml), streptomycin (6.5 µg/ml), and 5% fetal bovine serum (FBS). Cells were plated and incubated for 2 h at 37°C in a humidified CO2 (5%) incubator. The monolayers were washed with HHBSS three times to remove nonadherent cells, and monolayers were incubated overnight at 37°C in the above RPMI medium before study.

Measurement of the effects of insulin concentration and incubation time on GRP78 mRNA and GRP78 protein levels
Macrophages incubated overnight (4x106 cells/well in six-well plates) were washed with HHBSS, and a volume of RPMI-1640 medium containing the additions listed above was added. To one set of duplicate plates, increasing concentrations of insulin (0–40 nM) were added to the respective wells, and cells were incubated for 120 min at 37°C as above. To the second set of duplicate plates, 10 nM insulin was added to each well, and the cells were incubated for varying periods of time (0–180 min) as above. The reactions were terminated by aspirating the medium. The monolayers in both sets were washed once with the above RPMI-1640 medium followed by the addition of a volume of the same medium. One plate from each duplicate set was processed for mRNA determination, and the other was processed for Western blotting. Total RNA was extracted by a single-step method using an RNeasy® mini kit (Qiagen, Chatsworth, CA), according to the manufacturer’s instructions. Total RNA was reverse-transcribed with 1 µg RNA in a 20-µl reaction mixture, using Molony murine leukemia virus reverse transcriptase (200 units) and oligo(dt) as the primer for 1 h at 42°C. The resulting cDNA (5 µl) was used as a template, and a 225-base pair (bp) segment of the GRP78 cDNA was amplified using a 17-mer upstream primer (5'-CCACTTGGGCTATAGCA-3'), identical to positions corresponding to amino acids 332–338, and a 16-mer downstream primer (5'-ACCGCCTGACACCTGA-3'), complimentary to position 253–259 of the amino acids encoded in the GRP78 mRNA. A 302-bp segment of mouse ß-actin (constitutive internal control) cDNA was coamplified using a set of polymerase chain reaction (PCR) primers provided in an R&D Systems kit (Minneapolis, MN). Amplification was carried out in a Techne thermal cycler PHC for 28 cycles (one cycle: 94°C for 45 s, 60°C for 45 s, and 72°C for 45 s). PCR products were analyzed on a 1.2% (w/v) agarose/ethidium bromide gel, which was photographed, and the intensity of the individual GRP78 and ß-actin mRNA bands was quantified using a Storm 860 Phosphorimager® (Molecular Dynamics, marketed by Amersham Biosciences, Piscataway, NJ). Monolayers to be processed for Western blotting of GRP78 protein were lysed in a volume of lysis buffer containing 20 mM Tris-HCl (pH 8.6), 0.1 M NaCl, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, and 0.5% Nonidet for 10 min on ice. DNA strands were broken by passing the lysate through a 27-gauge needle and a syringe several times. The lysate was centrifuged at 800 g for 5 min at 4°C to remove cell debris. The supernatants were transferred to clean tubes, and their protein contents were determined [19 ]. Equal amounts of all lysate protein were used for electrophoresis as described previously [16 17 18 ]. Proteins from the gels (10%) were transferred to Hybond P® membrane (Amersham Biosciences) and immunoblotted with antibody against GRP78, according to the manufacturer’s instructions. GRP78 protein bands on the membrane were visualized by enhanced chemifluorescence (ECF) (Amersham Biosciences) and quantified using a Storm 860 Phosphorimager® (Amersham Biosciences). In preliminary, parallel experiments, under identical experimental conditions, the effect of time of incubation of cells with tunicamycin was also examined. The detection of GRP78 by Western blotting and quantification by ECF were performed as described above. All blots were reprobed for actin as a control.

Measurement of the effects of incubation time of cells with insulin on XBP-1, GADD153, and GADD34
Macrophages incubated overnight (4x106 cells/well in six-well plates) were washed with HHBSS and a volume of RPMI-1640 medium containing the additions listed. Insulin (10 nM) was added to cells, and the cells were incubated for varying periods of time (0–180 min) as above. The reactions were terminated by aspirating the medium. The monolayers were washed once with the RPMI-1640 medium followed by the addition of a volume of lysis buffer containing 20 mM Tris-HCl (pH 8.6), 0.1 M NaCl, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 20 µg/ml leupeptin, and 0.5% Nonidet for 10 min on ice. DNA strands were broken by passing the lysate through a 27-gauge needle and a syringe several times. The lysate was centrifuged at 800 g for 5 min at 4°C to remove cell debris. The supernatants were transferred to clean tubes, and their protein contents were determined [19 ]. Equal amounts of all lysate protein were used for electrophoresis as described previously. Proteins from the gels (10%) were transferred to Hybond P® membrane (Amersham Biosciences) and immunoblotted with antibody against XBP-1 and GADD153 or GADD34, respectively, according to the manufacturer’s instructions. XBP-1 and GADD protein bands on the membrane were visualized by ECF (Amersham Biosciences) and quantified as described above.

Measurements of signaling components and their activation in macrophages
Western blotting was used to measure proteins and/or their phosphorylation. PDK1, ERK1/2, p38 MAPK, PI-3K, JNK, Akt, p70 s6k, CREB, PERK, eIF2{alpha}, FKHR, 14-3-3, GSK3{alpha}/ß, XIAP, and Bcl-2 were all studied. For PI-3K, phosphorylation of the regulatory subunit was studied. For Akt, phosphorylation at Thr308 and Ser473 was investigated. Macrophages incubated overnight (4x106 cells/well in six-well plates) in RPMI-1640 medium, supplemented with 5% FBS, 2 mM glutamine, penicillin (12.5 units/ml), and streptomycin (6.5 µg/ml) in the respective wells, were exposed to buffer or insulin (10 nM/120 min). The reactions were terminated by aspirating the medium and lysing the cells in lysis buffer as described above. Other details of Western blotting and quantification of the respective protein bands on the membranes were as described above.

Determination of the effect of modulating insulin-induced, downstream signaling on the expression of GRP78 protein, p-PERK, and Bcl-2
Macrophages incubated overnight (4x106 cells/well in six-well plates) in RPMI-1640 medium supplemented with 5% FBS, 2 mM glutamine, penicillin (12.5 units/ml), and streptomycin (6.5 µg/ml) in the respective wells were exposed to one of the following: buffer; insulin (10 nM/120 min); PD 98059 (50 µM/120 min) then insulin (10 nM/120 min); SB203580 (15 µM/20 min) then insulin (10 nM/120 min); LY294002 (15 µM/20 min) then insulin (10 nM/120 min); and wortmannin (30 nM/30 min) then insulin (10 nM/120 min) [16 17 18 ]. The reactions were terminated at the end of the incubations by aspirating the medium and adding a volume of lysis buffer. Other details of electrophoresis, immunoblotting, and ECF visualization of immunoblots were identical to those described in the preceding sections. The respective membranes were reprobed for unphosphorylated eIF2{alpha} and for actin, according to the manufacturer’s instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of insulin on GRP78 mRNA and protein levels in macrophages
Transcription of the ER chaperone GRP78 is a classical marker of UPR activation in mammalian cells. To assess the induction of UPR as a result of increased global protein synthesis, we quantified the levels of GRP78 in cells exposed to physiological concentrations of insulin. The response was compared with that of cells exposed to tunicamycin, an agent routinely used to induce ER stress and UPR. Increasing insulin concentration and incubation time elevated GRP78 mRNA levels (Fig. 1A and 1B ). The effects of increasing insulin concentration and varying the incubation time on GRP78 mRNA nearly paralleled the effects on GRP78 protein levels (Fig. 1C and 1G) . In preliminary experiments, we compared the levels of insulin-induced up-regulation of GRP78 with GRP78 levels induced by tunicamycin (2.5 µg/ml). The kinetics of GRP78 expression in cells exposed to insulin compares fairly well with GRP78 expression in cells exposed to tunicamycin (Fig. 1E) . These results suggest that like tunicamycin, which has been widely used as an ER inducer, insulin also elevates the levels of GRP78, a biomarker of ER stress and UPR (Fig. 1E) . Therefore, in the following series of experiments, we have studied the effects of insulin only.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Effect of insulin on the expression of GRP78 and XBP-1. (A) Effect on GRP78 on mRNA levels of incubating cells (3 h) with varying concentrations of insulin. (B) Effect of time of incubation with insulin (10 nM) on mRNA levels of GRP78. (C) Effect of time of incubation of cells with insulin (10 nM) on GRP78 protein levels. (E) Effect of time of incubation with tunicamycin (2 µg/ml) on protein levels of GRP78. (G) Effect of incubating cells (3 h) with varying concentrations of insulin on GRP78 protein levels. (I) Effect of time of incubation of cells with insulin on protein levels of XBP-I. (D, F, H, and J) The respective protein-loading control actin. A representative immunoblot of respective protein components is shown. Three to five immunoblots from as many experiments were used for quantification of protein levels by phosphorimager (see Table 1 ).

 
Effects of insulin on XBP-1 protein in macrophages
In the following series of experiments, we analyzed the various components of UPR signaling. UPR is up-regulated by the ER transmembrane proteins IRE1, PERK, and ATF6. The kinase and RNase activities of mammalian IRE1{alpha}/ß are generated in response to the accumulation of unfolded proteins in the ER. The mRNA that encodes XBP-1, which interacts with the mammalian ER stress-response element, is the substrate for IRE1{alpha}/ß. On activation of UPR, IRE1{alpha}/ß cleaves XBP-1 mRNA to yield a potent transcriptional activator [5 6 7 8 9 10 11 12 13 ]. Transcription of XBP-1 is activated directly by UPR as well as by cleaved forms of ATF6, establishing a linkage between IRE1{alpha}/ß and ATF6 signaling to induce XBP-1 transcription and mRNA splicing [5 6 7 8 9 10 11 12 13 ]. The consequence of such an effect is that an increase in XBP-1 provides positive feedback for UPR activation. Physiological concentrations of insulin caused a transitory increase in XBP-1, which was maximal at ~30 min and declined after longer periods of incubation (Fig. 1I and Table 1 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Insulin and Expression of Components of UPR

 
Insulin activates PERK and eIF2{alpha}
All ER stresses that activate the transcriptional components of UPR also transiently attenuate protein synthesis, a response that is coupled to phosphorylation of PERK and eIF2{alpha}. We next determined levels of p-PERK and p-eIF2{alpha} and unphosphorylated eIF2{alpha} in cells exposed to insulin (10 nM) for varying periods of time (Fig. 2 and Table 1 ). Insulin-exposed cells demonstrated an approximate twofold increase in the levels of p-PERK (Fig. 2A and Table 1 ). The maximal effect of insulin was seen after 30–60 min exposure and declined at longer periods of incubation (Fig. 2A and Table 1 ). Insulin exposure also elevated the levels of p-eIF2{alpha} between 30 min and 60 min of incubation, which declined on longer periods of incubation (Fig. 2C) . Thus, it can be inferred that PERK-eIF2{alpha} signaling in insulin-treated cells permits an early recovery of protein synthesis.



View larger version (55K):
[in this window]
[in a new window]
  		Figure 2. Representative immunoblots showing the effect of time of incubation of cells with insulin on protein levels of p-PERK, p-eIF2{alpha}, and eIF2{alpha}; see Materials and Methods for details. Immunoblots are: (A) p-PERK; (B) actin protein-loading control for p-PERK; (C) p-eIF2{alpha} and (D) protein-loading control eIF2{alpha}; and (E) actin for p-eIF2{alpha}. Three to four immunoblots from as many experiments were used for quantification of protein levels by phosphorimager (see Table 1 ).

 
Insulin up-regulates GADD34
GADD34 is a member of the DNA damage and growth arrest-inducible gene family. Up-regulation of GADD34 requires the activation of PERK. GADD34 associates with protein phosphatase 1, which dephosphorylates eIF2{alpha}, and GADD34 is required for the robust expression of ER chaperones in response to ER stress. To understand the role of GADD34 in recovery from attenuated protein synthesis induced by phosphorylation of eIF2{alpha}, we determined the levels of GADD34 protein by Western blotting (Fig. 3A and Table 1 ). In insulin-exposed cells, the maximal expression of GADD34 occurred at 30 min of incubation but returned to basal values thereafter. These results suggest a rapid recovery from translational attenuation in insulin-induced ER stress.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 3. Representative immunoblots showing the effect of incubating macrophages with insulin on the protein levels of: (A) GADD34 and (C) GADD153. (B and D) Respective actin protein-loading controls are shown. Three to four immunoblots from as many experiments were used for quantification of protein levels by phosphorimager (see Table 1 ).

 
Expression of GADD153 in cells treated with insulin
We next examined the effect of insulin treatment on the expression of proapoptotic GADD153. Insulin-induced expression of GADD153 was modest compared with that of GADD34 (Fig. 3C) . These results indicate that insulin does not cause a major increase in the expression of proapoptotic GADD153 under our experimental conditions, unlike pharmacological inducers of ER stress [5 6 7 8 9 10 11 12 ].

PI-3K/Akt and MAPK signaling is involved in insulin-induced expression of PERK activation
One of the roles of UPR in ER stress is to protect cells from apoptosis. This is achieved by eliciting prosurvival and antiapoptotic signals. One of the best-characterized signaling cascades involved in these events is the PI-3K pathway. Binding of insulin to its cell-surface receptor activates PI-3K, which generates phosphatidylinositol [3 4 5 ]-triphosphate and phosphatidylinositol [3 , 4 ]-bisphosphate. These phospholipids recruit PDK1 and Akt to the plasma membrane, where full activation of Akt requires its phosphorylation at Ser473 and Thr308. PDK1 phosphorylates Akt at Thr308; however, the identity of the kinase responsible for Ser473 phosphorylation is not fully established. The effect of time of incubation of macrophages with insulin (10 nM) on the protein levels of Akt phosphorylated at Thr308 and Ser473 is shown in Figure 4 . Insulin treatment of cells elevated phosphorylation at both residues. Insulin treatment of cells increased PDK1 expression and the phosphorylation of protein levels of ERK1/2, p38 MAPK, the 85 kDa regulatory subunit of PI-3K, and p70 s6k by several-fold compared with buffer-treated controls (Fig. 4A) . However, the largest increase, nine- to 12-fold, was observed in the activation of JNK and Akt. Increases in p-PERK were also greatly reduced by genestein, PD98059, SB204583, LY294002, and wortmannin (Fig. 4B and Table 2 ), suggesting the possible role of MAPK and PI-3K in the activation of PERK under ER stress. Activation of PERK requires its phosphorylation. However, the protein kinase(s) involved in PERK phosphorylation has not been identified. As inhibition of the activation of MAPK and PI-3K signaling reduced the levels of p-PERK, it is concluded that the kinases of these signaling cascades participate in the activation of PERK directly or indirectly. These data also suggest that PERK is activated by upstream signaling events.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. Representative immunoblots showing the effect of insulin on the activation of protein kinases and the modulation of PERK activation. Immunoblots are: (A) phosphorylation of ERK1/2 (1); p38 MAPK (2); JNK (3); 85 kDa regulation subunits of PI-3K (4); PDK1 (5); Akt phosphorylated at Thr308 (6); Akt phosphorylated at Ser473 (7); and p-70S6k (8) in buffer (a) and insulin-treated cells (b). Insulin induced activation of these protein kinases compared with the vehicle control (a); represented as bar diagram above the immunoblot (b). Values are expressed in arbitrary units as mean ± SE from three experiments. (B) Modulation insulin-induced activation of PERK by MAPK and PI-3K. A representative immunoblot of PERK and its loading control actin is shown. Three to four immunoblots from as many experiments were used for quantification of PERK protein levels by phosphorimager (see Table 2 ). The lanes are: (1) buffer; (2) insulin (10 nM/90 min); (3) PD98059 (50 µM/90 min) then insulin; (4) SB203580 (20 µM/20 min) then insulin; (5) LY294002 (20 µM/20 min) then insulin; and (6) wortmannin (30 nM/30 min) then insulin.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Effect of Pharmacological Interventions on Activation of Signaling Molecules

 
Phosphorylation of FKHR in insulin-exposed cells
Akt regulates cell survival by directly phosphorylating components of the cell death apparatus; namely, Bad, caspase 9, and forkhead family members such as FKHR [20 ]. Phosphorylation of FKHR by activated Akt promotes its export from the nucleus to the cytosol and thus prevents FKHR interaction with DNA and up-regulation of transcription factors involved in the apoptotic pathway [21 , 22 ]. FKHR also interacts with the 14-3-3 protein, which serves to localize the p-FKHR in the cytoplasm [23 , 24 ]. Binding of 14-3-3 to p-FKHR also facilitates nuclear export of FKHR in insulin-treated cells [21 22 23 24 ]. Insulin treatment of cells profoundly elevated the levels of phosphorylated FKHR at 30–60 min post-insulin treatment, and it declined on longer periods of incubation (Fig. 5 and Table 3 ). Insulin also elevated the levels of 14-3-3 protein, thereby showing its role in regulation of FKHR localization (Fig. 5) . These results show that the prosurvival effects of insulin are mediated by Akt, which phosphorylates nuclear FKHR and thus prevents FKHR-mediated expression of proapoptotic genes under our experimental conditions.



View larger version (82K):
[in this window]
[in a new window]
  		Figure 5. Representative immunoblots showing the effect of time of incubation of cells with insulin on the levels of: (A) p-FKHR; (B) FKHR; (C) 14-3-3; (D) actin, used as a protein-loading control for 14-3-3; (E) GSK3{alpha}/ß; (F) actin protein-loading control for GSK3{alpha}/ß; (G) XIAP; (H) actin protein-loading control for XIAP. Three immunoblots from as many experiments were used for quantification of protein levels by phosphorimager (see Table 3 ).

 

View this table:
[in this window]
[in a new window]
  		Table 3. Insulin and Antiapoptotic Signaling Cascades

 
Inhibition of GSK3 in cells exposed to insulin
GSK3{alpha}/ß is a serine/threonine kinase, which phosphorylates glycogen synthase, causing its inactivation [25 , 26 ]. Phosphorylation of GSK3{alpha} at Ser21 and GSK3ß at Ser9 by several kinases including Akt, protein kinase A (PKA), PKC, and p70 s6k results in its inactivation. We have assayed the levels of p-GSK3ß in cells treated with insulin to understand the part played by GSK3{alpha}/ß in cell survival under our experimental conditions (Fig. 5 and Table 3 ). Exposure of cells to insulin caused maximal phosphorylation of GSK3ß at a 60- to 180-min period, which remained elevated up to 3 h of incubation (Fig. 5 and Table 3 ). The results suggest an Akt-mediated inactivation of GSK3{alpha}/ß to prevent it from activating apoptotic pathways.

Effect of insulin on XIAP
IAP is a family of proteins that regulates cell death (ref. [27 ] and references therein). XIAP blocks mitochondrial- and death receptor-mediated pathways of apoptosis by binding directly to and inhibiting initiator and effector caspases. In this study, we have determined the levels of XIAP in insulin-treated cells to understand its antiapoptotic role in insulin-induced ER stress (Fig. 5 and Table 3 ). Exposure of cells to insulin elevated the levels of XIAP, suggesting the antiapoptotic role of XIAP proteins in insulin-induced ER stress and recovery from it through UPR (Fig. 5 and Table 3 ).

Insulin-induced activation of CREB and Bcl-2
Up-regulation of Bcl-2 expression has been identified as a critical mechanism for promotion of cell survival. The promoter region of the antiapoptotic factor Bcl-2 contains a cAMP response element site, and the transcription factor CREB, when activated, is a positive regulator of Bcl-2 [28 , 29 ]. Akt has been shown to activate CREB. Activation of CREB is also PI-3K-dependent; thus, insulin should up-regulate Bcl-2 activation, and indeed, this is correct. Insulin caused a maximal increase in the levels of p-CREB at ~30 min of incubation, which persisted 2–3 h (Fig. 6 and Table 3 ). Exposure of cells to 10 nM insulin for 2 h elevated the levels of p-Bcl-2 by approximately twofold compared with buffer-treated cells (Fig. 6 and Table 2 ). Pretreatment of cells with SB203580, LY294002, H-89, or PD98059, the inhibitors of p38 MAPK, PI-3K, PKA, and ERK1/2, respectively, before insulin exposure drastically reduced insulin-induced, elevated levels of Bc1-2 (Fig. 6 and Table 2 ). These studies demonstrate that CREB is not only involved in insulin-induced GRP78 expression but also activates Bcl-2, an event correlated with protection against apoptosis.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 6. Representative immunoblots showing the effect of time of incubation on: (A) phosphorylation of CREB (p-CREB) Ser133; (B) protein levels of CREB; (C) modulation of insulin up-regulated protein levels of Bcl-2 by MAPK, PI-3K, and PKA. The lanes are: (1) buffer; (2) insulin (10 nM/90 min); (3) PD98059 (50 µM/90 min) then insulin; (4) SB203580 (20 µM/20 min) then insulin; (5) LY294002 (20 µM/20 min) then insulin; and (6) H-89 (15 µM/90 min) then insulin. (D) Actin protein-loading control for Bcl-2. Three to four immunoblots from as many experiments were used for quantification of protein levels by phosphorimager (see Tables 2 and 3 ).

 
A schematic representation of the activation of UPR by ER stress induced by insulin is depicted in Figure 7 .



View larger version (36K):
[in this window]
[in a new window]
  		Figure 7. A schematic representation of the activation of UPR in macrophages treated with insulin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The salient observations of this study are that treatment of peritoneal macrophages with insulin results in the induction of ER stress, as evidenced by elevated expression of GRP78; the cells protect themselves against adverse effects of ER stress by triggering UPR, as evidenced by elevated expression of XBP1, p-PERK, p-eIF2{alpha}, and GADD34; and cells also protect themselves by elevating the expression of antiapoptopic cascades, as evidenced by Bcl-2, p-FKHR, GSK3{alpha}/ß, XIAP, and p-CREB at Ser133.

The exposure of eukaryotic cells to pharmacological agents such as tunicamycin as well as certain pathophysiological conditions causes perturbations in ER functions, which generate ER stress (see refs. [5 6 7 8 9 10 11 12 13 ]). Mammalian cells protect themselves from many effects of ER stress by triggering UPR [5 6 7 8 9 10 11 12 13 ]. As unfolded proteins accumulate, UPR coordinates a broad down-regulation of protein synthesis with increased expression of various gene products including ER resident molecular chaperones that promote protein folding. The homology displayed among lumenal domains of PERK, IRE1{alpha}, and IRE1ß suggests that they use a similar mechanism to transduce ER stress. IRE1{alpha} may sense the levels of free GRP78 in the ER so that in the absence of unfolded proteins, GRP78 binds to IRE1 and prevents its oligomerization and subsequent activation. On accumulation of unfolded proteins in the ER, GRP78 oligomers dissociate and trigger. Recent reports demonstrate that activation of UPR can be induced in normal, cellular, developmental processes, which increase the demand on the protein-folding capacity of the ER [30 31 32 ]. Activation of major components of UPR such as XBP-1, GRP78, GRP94, and ATF6{alpha} occurs in antibody-secreting plasma cells [30 31 32 33 34 35 36 ]. Stimulation of splenocytes with lipopolysaccharide resulted in the expression of XBP-1 [30 31 32 33 ]. Such stimulation in B cells also results in the synthesis of GRP78 and GRP94 [35 , 36 ]. However, the mechanism of activation of UPR in antibody-synthesizing cells is different than that induced by agents such as tunicamycin, as there was minimal expression of proapoptotic GADD153 [30 ]. Here, we demonstrate that physiological concentrations of insulin induce ER stress and activate UPR in peritoneal macrophages with minimal expression of proapoptopic GADD153, which suggests that under these conditions, the major function of UPR is the protection of cells from apoptotic processes.

The protective effect of UPR against ER stress in insulin-treated cells is supplemented by the activation of the PI-3K and MAPK signaling cascades induced upon binding of insulin to its receptors on the cell surface. Binding of insulin to cell-surface receptors results in the alterations in the expression of many genes for cellular growth, differentiation, and proliferation [37 ]. A series of genes has been identified that controls developmental cell death. Bcl-2 is the prototype for a large family of structurally related proteins that regulates cell death in mammalian cells [20 ]. Some of these proteins, such as Bcl-2 and Bcl-XL, promote cell survival, whereas other proteins, such as Bax and Bad, promote cell death. Akt affects its antiapoptotic activity by phosphorylating Bad [38 ]. The promoter region of the antiapoptotic factor Bcl-2 contains a cAMP response element site, and the transcription factor CREB, when activated by multiple kinases, is a positive regulator of Bcl-2 [28 , 29 ]. Elevated expression of activated CREB and Bcl-2 in insulin-treated macrophages suggests their involvement in cell survival under the experimental conditions. The effect of insulin on cellular growth, differentiation, and proliferation is mediated by its downstream effector Akt. Several-fold increases in the activation of JNK and Akt phosphorylated at Ser473 in cells exposed to insulin demonstrate that insulin-activated PI-3K/Akt downstream signaling plays a critical antiapoptotic and survival role in insulin-induced ER stress and UPR. Akt may promote cell survival by directly phosphorylating transcription factors that control the expression of pro- and antiapoptotic genes, namely members of the forkhead transcription factors. In some cell types, Akt increases the expression of the antiapoptotic gene Bcl-2. Induction of Bcl-2 promoter activity by insulin-growth factor 1 occurs via the Akt pathway and involves CREB, which is phosphorylated directly by Akt. This phosphorylation increases binding of CREB to accessory proteins necessary for induction of genes containing CRE elements in their promoter region [39 , 40 ]. Akt also phosphorylates inhibitor of {kappa}B and thus promotes its degradation and consequently, the translocation of nuclear factor (NF)-{kappa}B to the nucleus, where it activates the prosurvival gene IAP [20 , 39 , 40 ]. NF-{kappa}B is involved in the regulation of cell proliferation, apoptosis, and survival by a wide range of cytokines and growth factors. Akt also promotes survival by directly phosphorylating regulators of the apoptotic cascade. Stress-activated protein kinases such as JNK are critically involved in the induction of apoptosis following exposure of cells to ionizing radiation, heat shock, or osmotic stress [41 ]. Akt phosphorylates and inactivates apoptosis signal-regulating kinase-1, a kinase that transduces stress signals through the JNK and p38 MAPK pathways and thus inhibits apoptosis [42 ]. In this report, we further show the requirement of MAPK and PI-3K/Akt signaling in the expression of components of the UPR signaling cascade, namely, GRP78, PERK, and Bcl-2.

In conclusion, we show that treatment of peritoneal macrophages with insulin (10 nM) triggers ER stress as a result of an imbalance between protein synthesis and protein-folding capacity of ER. The system senses ER stress through its ER stress sensors and initiates UPR signaling by activating IRE1 and PERK to protect cells from the adverse effects of ER stress. Concomitantly, signaling downstream to receptor ligation protects cells from apoptosis by up-regulating the PI-3K/Akt/CREB signaling pathways.


   ACKNOWLEDGEMENTS
 
This work was supported by Grant HL-24066 from the National Heart, Lung, and Blood Institute.

Received November 23, 2004; revised March 2, 2005; accepted March 29, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nicholson, K. M., Anderson, N. G. (2002) The protein kinse B/Akt signaling pathway in human malignancy Cell. Signal. 14,381-395[CrossRef][Medline]
  2. Downward, J. (2004) PI 3-kinase, Akt and cell survival Semin. Cell Dev. Biol. 15,177-182[CrossRef][Medline]
  3. Hanada, M., Feng, J., Hemmings, B. A. (2004) Structure, regulation and function of PKB/Akt—a major therapeutic target Biochim. Biophys. Acta 1697,3-16[Medline]
  4. Proud, C. G., Denton, R. M. (1997) Molecular mechanisms for the control of translation by insulin Biochem. J. 328,329-341
  5. Rutkowski, D. T., Kaufman, R. J. (2004) A trip to the ER: coping with stress Trends Cell Biol. 14,20-28[CrossRef][Medline]
  6. Kaufman, R. J. (2004) Regulation of mRNA translation by protein folding in the endoplasmic reticulum Trends Biochem. Sci. 29,152-158[CrossRef][Medline]
  7. Zhang, K., Kaufman, R. J. (2004) Signaling the unfolded protein response from the endoplasmic reticulum J. Biol. Chem. 279,25935-25938[Free Full Text]
  8. Lee, A. S. (2001) The glucose-regulated proteins: stress induction and clinical applications Trends Biochem. Sci. 26,504-510[CrossRef][Medline]
  9. Bertolotti, A., Zhang, Y., Hendershot, L. M., Harding, H. P., Ron, D. (2000) Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response Nat. Cell Biol. 2,326-332[CrossRef][Medline]
  10. Kaufman, R., Scheunder, D., Schroder, M., Shen, X., Lee, K., Liu, C. Y., Arnold, S. M. (2002) The unfolded protein response in nutrient sensing and differentiation Nat. Rev. Mol. Cell Biol. 3,411-421[CrossRef][Medline]
  11. Harding, H. P., Calfon, M., Urano, F., Novoa, I., Ron, D. (2002) Transcriptional and translational control in the mammalian unfolded protein response Annu. Rev. Cell Dev. Biol. 18,575-599[CrossRef][Medline]
  12. Haze, K., Yoshida, H., Yura, T., Mori, K. (1999) Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress Mol. Biol. Cell 10,3787-3799[Abstract/Free Full Text]
  13. Liu, C. Y., Schroder, M., Kaufman, R. J. (2000) Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of endoplasmic reticulum J. Biol. Chem. 275,24881-24885[Abstract/Free Full Text]
  14. Rao, R. V., Peel, A., Logvinova, A., delRio, G., Hermel, E., Yokota, T., Goldsmith, P. C., Ellerby, L. M., Ellerby, H. M., Bredesen, D. E. (2002) Coupling endoplasmic reticulum stress to cell death program: role of ER chaperone GRP78 FEBS Lett. 514,122-128[Medline]
  15. Reddy, R. K., Mao, C., Baumeister, P., Austin, R. C., Kaufman, R. J., Lee, A. S. (2003) Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors. Role of ATP binding site in suppression of caspase-7 activation J. Biol. Chem. 278,20915-20924[Abstract/Free Full Text]
  16. Misra, U. K., Pizzo, S. V. (2002) Regulation of cytosolic phospholipase A2 activity in macrophages stimulated with receptor-recognized forms of {alpha}2-macroglobulin: role in mitogenesis and cell proliferation J. Biol. Chem. 277,4069-4078[Abstract/Free Full Text]
  17. Misra, U. K., Akabani, G., Pizzo, S. V. (2002) The role of cAMP-dependent signaling in receptor-recognized forms of {alpha}2-macroglobulin-induced cellular proliferation J. Biol. Chem. 277,36509-36520[Abstract/Free Full Text]
  18. Misra, U. K., Gonzalez-Gronow, M., Gawdi, G., Hart, J. P., Johnson, C. E., Pizzo, S. V. (2002) The role of Grp 78 in {alpha}2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction J. Biol. Chem. 277,42082-42087[Abstract/Free Full Text]
  19. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 72,248-254[CrossRef][Medline]
  20. Datta, S. R., Brunet, A., Greenberg, M. D. (1999) Cellular survival: a play in three Akts Genes Dev. 13,2905-2927[Free Full Text]
  21. van Der Heide, L. P., Hoekman, M. F., Smidt, M. P. (2004) The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation Biochem. J. 380,297-309[CrossRef][Medline]
  22. Burgering, B. M., Kops, G. J. P. L. (2002) Cell cycle and death control: longlive forkheads Trends Biochem. Sci. 27,352-360[CrossRef][Medline]
  23. Mackintosh, C. (2004) Dynamic interactions between 14-3-3 proteins and phospho-proteins regulate diverse cellular processes Biochem. J. 381,329-342[CrossRef][Medline]
  24. Tzivion, G., Avruch, J. (2002) 14-3-3 Proteins: active cofactors in cellular regulation by serine/threonine phosphorylation J. Biol. Chem. 277,3061-3064[Free Full Text]
  25. Kaytor, M. D., Orr, H. T. (2002) The GSK3ß signaling cascade and neurodegenerative disease Curr. Opin. Neurobiol. 12,275-278[CrossRef][Medline]
  26. Jope, R. S., Johnson, G. V. (2004) The glamour and gloom of glycogen synthase kinase 3 Trends Biochem. Sci. 29,95-102[CrossRef][Medline]
  27. Holcik, M., Korneluk, R. G. (2001) XIAP, the guardian angle Nat. Rev. Mol. Cell Biol. 2,550-556[CrossRef][Medline]
  28. Pugazhenthi, S., Miller, E., Sable, C., Heidenreich, K. A., Boxer, L. M., Reusch, J. E. B. (1999) Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein J. Biol. Chem. 274,27529-27535[Abstract/Free Full Text]
  29. Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K. A., Boxer, L. M., Heasley, L. E., Reusch, J. E-B. (2000) Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein J. Biol. Chem. 275,10761-10768[Abstract/Free Full Text]
  30. Gass, J. N., Gifford, N. M., Brewer, J. W. (2002) Activation of an unfolded protein response during differentiation of antibody-secreting B cells J. Biol. Chem. 277,49047-49054[Abstract/Free Full Text]
  31. Ma, Y., Brewer, J. W., Diehl, J. A., Hendershot, L. M. (2002) Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response J. Mol. Biol. 318,1351-1365[CrossRef][Medline]
  32. Brewer, J. W., Cleveland, J. L., Hendershot, L. M. (1997) A pathway distinct from the mammalian unfolded protein response regulates expression of endoplasmic reticulum chaperones in non-stressed cells EMBO J. 16,7207-7216[CrossRef][Medline]
  33. Calfon, M., Zeng, H., Urano, F., Till, J. H., Hubbard, S. R., Harding, H. P., Clark, S. G., Ron, D. (2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA Nature 415,92-96[CrossRef][Medline]
  34. Reimold, A. M., Iwakoshi, N. N., Manis, J., Vallbhajosyula, P., Szomolanyi-Tsuda, E., Gravallese, E. M., Friend, D., Grusby, M. J., Alt, F., Glimcher, L. H. (2001) Plasma cell differentiation requires the transcription factor XBP-1 Nature 412,300-307[CrossRef][Medline]
  35. Wiest, D. L., Burkhardt, J. K., Hester, S., Hortsch, M., Meyer, D. I., Argon, Y. (1990) Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately J. Cell Biol. 110,1501-1511[Abstract/Free Full Text]
  36. Lewis, M. J., Mazzarella, R. A., Green, M. (1985) Structure and assembly of the endoplasmic reticulum. The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes J. Biol. Chem. 260,3050-3057[Abstract/Free Full Text]
  37. Shepherd, P. R., Withers, D. J., Siddle, K. (1998) Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling Biochem. J. 333,471-490
  38. Cichy, S. B., Uddin, S., Danilkovich, A., Guo, S., Klipple, A., Unterman, T. G. (1998) Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence J. Biol. Chem. 273,6482-6487[Abstract/Free Full Text]
  39. Nicholson, K. M., Anderson, N. G. (2002) The protein kinase B/Akt signaling pathways in human malignancy Cell. Signal. 14,381-395
  40. Du, K., Montiminy, M. (1998) CREB is a regulatory target for the protein kinase Akt/PKB J. Biol. Chem. 273,32377-32379[Abstract/Free Full Text]
  41. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., Donner, D. B. (1999) NF-{kappa}B activation by tumor necrosis factor requires the Akt serine-threonine kinase Nature 401,82-85[CrossRef][Medline]
  42. Kim, A. H., Khursigara, G., Sun, X., Frank, T. F., Chao, M. V. (2001) Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1 Mol. Cell. Biol. 21,893-901[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Gonzalez-Gronow, S. J. Kaczowka, S. Payne, F. Wang, G. Gawdi, and S. V. Pizzo
Plasminogen Structural Domains Exhibit Different Functions When Associated with Cell Surface GRP78 or the Voltage-dependent Anion Channel
J. Biol. Chem., November 9, 2007; 282(45): 32811 - 32820.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. J. Pagliassotti, Y. Wei, and D. Wang
Insulin Protects Liver Cells from Saturated Fatty Acid-Induced Apoptosis via Inhibition of c-Jun NH2 Terminal Kinase Activity
Endocrinology, July 1, 2007; 148(7): 3338 - 3345.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
U. K. Misra, R. Deedwania, and S. V. Pizzo
Activation and Cross-talk between Akt, NF-{kappa}B, and Unfolded Protein Response Signaling in 1-LN Prostate Cancer Cells Consequent to Ligation of Cell Surface-associated GRP78
J. Biol. Chem., May 12, 2006; 281(19): 13694 - 13707.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1104685v1
78/1/187    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Misra, U. K.
Right arrow Articles by Pizzo, S. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Misra, U. K.
Right arrow Articles by Pizzo, S. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS