JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404280200 on July 12, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39348-39357, September 17, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/38/39348    most recent
M404280200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 Balbis, A.
Right arrow Articles by Posner, B. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Balbis, A.
Right arrow Articles by Posner, B. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Effect of Insulin on Caveolin-enriched Membrane Domains in Rat Liver*

Alejandro Balbis, Gerardo Baquiran, Catherine Mounier, and Barry I. Posner{ddagger}

From the Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, 3640 University St., Suite W315, Montreal, Quebec H3A 2B2, Canada

Received for publication, April 16, 2004 , and in revised form, June 25, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Compartmentalization of signaling molecules may explain, at least in part, how insulin or growth factors achieve specificity. Caveolae/rafts are specialized lipid compartments that have been implicated in insulin signaling. In the present study, we investigated the role of caveolin-enriched membrane domains (CMD) in mediating insulin signaling in rat liver. We report the existence of at least two different populations of CMD in rat liver plasma membranes (PM). One population is soluble in Triton X-100 and seems to be constitutively associated with cytoskeletal elements. The other population of CMD is located in a membrane compartment insoluble in Triton X-100 with light buoyant density and is hence designated CMD/rafts. We found evidence of rapid actin reorganization in rat liver PM in response to insulin, along with the association of CMD/rafts and insulin signaling molecules with a cell fraction enriched in cytoskeletal elements. The presence of CMD in liver parenchyma cells was confirmed by the presence of caveolin-1 in primary rat hepatocyte cultures. Cholesterol depletion, effected by incubating hepatocytes with 2 mM methyl-{beta}-cyclodextrin, did not permeabilize the cells or interfere with clathrin-dependent internalization. However, at this concentration, methyl-{beta}-cyclodextrin perturbed CMD of hepatocyte PM and inhibited insulin-induced Akt activation and glycogen synthesis but did not affect insulin-induced insulin receptor kinase tyrosine phosphorylation. These events, together with the presence of a functional insulin receptor in CMD of rat liver PM, suggest that insulin signaling is influenced by the interaction of caveolae with cytoskeletal elements in liver.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although binding of insulin to its receptor is a process specific to high affinity, the signaling cascades generated by the activated insulin receptor kinase (IRK)1 are shared by a number of other growth factors (1–3). Nevertheless, the metabolic actions of insulin cannot be reproduced in intensity and quality by other hormones or growth factors. The mechanism by which insulin achieves its specificity of action remains to be satisfactorily determined. Compartmentalization of signaling molecules in plasma membranes and endosomes may play an important role in determining the specificity of signal transduction (4–7).

The recent isolation of detergent-insoluble, low-density membrane fragments from cells suggests that sphingolipid and cholesterol rich domains could exist as a liquid-ordered phase in the membrane (8). These lipid domains, known as lipid rafts, can recruit or exclude signaling proteins. Thus, lipid rafts have been implicated in the regulation of hormone and growth factor signaling (8, 9). Caveolae, which constitute a subset of lipid rafts, are invaginated cell surface microdomains that are enriched in caveolin oligomers, the major protein constituent of these structures (10, 11). Three caveolins (caveolin-1, -2, and -3) have been discovered. Caveolin-1 and caveolin-2 are found most abundantly in adipocyte and endothelial cells, whereas caveolin-3 is found in muscle cells. Caveolae have been implicated in potocytosis, transcytosis, endocytosis independent of clathrin, and signal transduction (11, 12). It has been shown that caveolae negatively affect epidermal growth factor and Src signaling (13). In contrast to these inhibitory effects, a number of studies, mostly in adipocytes, have suggested that intact caveolae are necessary for insulin signaling. In 3T3-L1 adipocytes, IRK was reported to be concentrated in caveolae (14) and to interact with caveolin-1 to modulate insulin signaling (15, 16). It has also been reported that insulin induced tyrosine phosphorylation of caveolin (17). Disrupting the lipid structure of caveolae by depleting their cholesterol content with {beta}-cyclodextrin attenuated IRK signaling (14, 18). Rafts/caveolae have also been implicated in insulin-stimulated glucose transport in 3T3-L1 adipocytes by a mechanism independent of PI3-kinase (19, 20). Caveolin-1 knockout mice have been created that are viable despite a complete ablation of caveolae (21). These mice show impaired nitric oxide signaling, vascular dysfunction, hyperproliferation of endothelial cells, abnormalities in lipid homeostasis, insulin resistance, and decreased insulin receptor expression in adipose tissue (21–23). These observations suggest that caveolae in adipocytes can contribute to both the strength and specificity of insulin signaling.

There is a paucity of data concerning the role of caveolae on insulin signaling in other insulin-responsive tissues. Although liver contains a lower level of caveolin-1 than that in adipocytes, it has nevertheless clearly been shown that caveolin-1 is located in liver parenchymal cells, with negligible levels detected in endothelial cells (24, 25). Furthermore, caveolae have been demonstrated at the cell surface of hepatocytes using rapid-freeze, deep-etching electron microscopy (25). Because liver is an important insulin target tissue, we investigated the significance of caveolin-enriched membrane domains (CMD) in mediating insulin signaling in rat liver. In the present study, we have characterized these entities and have demonstrated a possible function in insulin signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Female Sprague-Dawley rats, 10 weeks of age, (160–180 g body weight) were purchased from Charles River Laboratories Canada Ltd. (St. Constant, PQ, Canada), housed in an animal facility with 12-h light cycles at 25 °C and fed ad libitum on Purina chow. Animals were fasted overnight (16–18 h) before each study.

Materials—Porcine insulin was a gift from Eli Lilly and Co., (Indianapolis, IN). Phenylmethylsulfonyl fluoride, sodium orthovanadate, methyl-{beta}-cyclodextrin, and most other chemicals were purchased from Sigma. Reagents for electrophoresis were from Bio-Rad. Kodak X-OMAT AR film was from Picker International (Montreal, PQ, Canada). Polyvinylidene difluoride Immobilon-P transfer membranes were from Millipore Ltd. (Mississauga, ON, Canada). [U-14C]glucose (300 mCi/mmol) was purchased from PerkinElmer Life and Analytical Sciences. Magnetic beads (Dynabeads M-280) were from Dynal (Lake Success, NY). An antibody raised against a peptide corresponding to residues 942–969 of the juxtamembrane region of the IRK {beta}-subunit (anti-960) was prepared and purified on a protein A-Sepharose column as described previously (26) and used for Western blotting. For immunoprecipitation of IRK, an antibody directed against the {alpha}-subunit was obtained from the serum of a patient with acanthosis nigricans (27). Polyclonal anti-p85, a polyclonal anti-IRS1 antibody, and a specific antibody against Akt2 were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Antibodies against Akt1 and phospho-Akt1 (Ser473) were purchased from New England Biolabs, Inc. (Mississauga, ON, Canada). An antibody against actin and iron-saturated transferrin was purchased from Sigma (St. Louis, MO). A monoclonal anti-phosphotyrosine antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caveolin-1 was purchased from BD Transduction Laboratories.

Preparation of Subcellular Fractions—Rats were anesthetized and sacrificed by decapitation at the indicated times after intrajugular injections as described in the appropriate figure legends. Livers were exsanguinated, rapidly excised, and minced at scissor-point in ice-cold buffer (5 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl2, 2 mM NaF, and 2 mM Na3VO4). Plasma membranes (PM), endosomes, and microsomes were prepared as described previously (27). A purified Golgi fraction prepared as described previously (28) was kindly provided by Dr. Bergeron. The protein content of these fractions was measured using a modification of Bradford's method with bovine serum albumin as standard (29).

Isolation of Caveolin-enriched Membrane Domains (CMD)—Isolation of CMD with Triton X-100 was accomplished as follows. CMD were isolated by a modification of the method of Liu et al. (30). In brief, plasma membranes were pelleted and mixed with 3 ml of ice-cold 1% Triton-X100 in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4). The samples were homogenized (10 strokes in a glass homogenizer), incubated on ice for 1 h, adjusted to the same amount of protein, and diluted 1:1 with 80% sucrose in buffer B (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). The extract (4 ml, between 2 and 5 mg of protein) was loaded on the bottom of a 12-ml ultracentrifuge tube and overlaid with 4 ml each of 30 and 10% sucrose in buffer B. The gradient was centrifuged for 21 h at 29,000 x g in a SW40 Ti rotor (Beckman Instruments), and 1-ml fractions were collected from the top of the tube. Fractions 1–8 (10–30% sucrose gradient), fraction 9 (soluble proteins in the residual 40% sucrose layer), and the pellet, re-suspended in 1 ml of ice cold phosphate buffered saline (PBS), were subsequently analyzed by SDS-PAGE and Western blotting. The amount of protein recovered in the detergent-resistant membranes (DRM)/lipid raft fraction (fractions 4 and 5 of sucrose gradient) and in the Triton-insoluble pellet was between 20 and 100 µg and 400 and 700 µg, respectively. Alternatively, CMD was isolated with a detergent-free method. In brief, PM was mixed with 3 ml of ice-cold Na2CO3 (200 mM, pH 11) in buffer A. Samples were homogenized (10 strokes in a glass homogenizer), sonicated (3 times, 10 s each), and incubated on ice for 1 h. Thereafter, samples were mixed with 80% sucrose, centrifuged overnight, and collected as indicated above. Finally, in some experiments, PM were homogenized in 1% Triton X-100 and Na2CO3 (pH 11; final concentration, 200 mM) in buffer A. After incubating on ice for 1 h, the samples were mixed with an equal volume of 80% sucrose (1:1) and subjected to sucrose gradient centrifugation as described above.

Immunoblotting—Fractions 1–8 (100 µl each) from the sucrose gradients were mixed with 50 µlof3x Laemmli sample buffer subjected to SDS-PAGE (6–12% gel) and then transferred to Immobilon-P membranes for immunoblotting. In some experiments, proteins from these fractions were concentrated with trichloroacetic acid (30) and dissolved in 100 µl of 1x Laemmli buffer. The Triton-insoluble pellet was resuspended in 1 ml of PBS buffer, mixed with 500 µl of 3x Laemmli sample buffer, and aliquots of 100 µl were used for SDS-PAGE. 50 µgof protein from fraction 9 and total PM were used for SDS-PAGE. Immunoblotting with anti-caveolin-1 antibody showed a nonspecific band at 29 kDa when total membranes or fraction 9 were analyzed. This band was absent in the Triton-insoluble pellet and in lipid rafts. Either I125GAR or I125GAM were used as secondary antibodies and, after autoradiography at –80 °C, appropriate bands were quantified using a Bio-Rad GS-700 imaging densitometer.

Immunoisolation of CMDs—PM was homogenized in Na2CO3, pH11 (10 strokes in a Dounce homogenizer), and incubated on ice for 1 h followed by centrifugation at 200,000 x g for 1 h. The resultant pellet was again homogenized and then sonicated (three times, 10 s each) in Tris-buffered saline (buffer B) containing proteases inhibitors and 1% albumin as noted above. The treatment with Na2CO3 was carried out to remove PM filaments, which could interfere with the immunoisolation process. Magnetic beads (Dynabeads M-280) were coated with a specific antibody against caveolin-1 or with IgG (negative control) as specified by the manufacturer. Coated beads were incubated with PM for 2 h at 4 °C, resuspend and washed four times with Tris-buffered saline, pH 7.5, before boiling for 5 min in Laemmli sample buffer, and subjecting to SDS-PAGE.

Cell Culture—Primary hepatocytes, isolated from 120–140-g male Sprague-Dawley rats (Charles River Laboratories, Inc.) by in situ liver perfusion with collagenase, were plated on a collagen matrix (Vitrogen-100). Subconfluent cultures were prepared by seeding 1 x 106 cells, onto 9.6-cm2 6-well plates (Corning Costar, Cambridge, MA) or 5 x 106 cells, onto 78 cm2 culture dishes (Starstedt Canada, St. Laurent, PQ, Canada). Cells were bathed for 24 h in seeding medium (Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% fetal bovine serum, 10 mM HEPES, 20 mM NaHCO3, 500 IU/ml penicillin, and 500 µg/ml streptomycin) and then for 48 h in serum-free medium that differed from the seeding medium in that it lacked FBS and contained 1.25 µg/ml Fungizone, 0.4 mM ornithine, 2.25 µg/ml L-lactic acid, 2.5 x 10–8 M selenium, and 1 x 10–8 M ethanolamine. Fresh serum-free media was added just before incubation of hepatocytes with methyl-{beta}-cyclodextrin (M{beta}CD).

Biotinylation of Cell Surface Proteins—Detection of IRK located at the cell surface of the hepatocytes was performed as described previously (31). In brief, hepatocytes were incubated in the presence or absence of M{beta}CD and insulin as noted in the figure legends. Thereafter, hepatocytes were washed three times with ice-cold PBS-Ca-Mg, pH 7.4 (0.1 mM CaCl2 and 1 mM MgCl2), and cell surface proteins were biotinylated by incubation with 0.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) in PBS-Ca-Mg for 30 min at 4 °C. The reaction was stopped by washing the dishes three times with PBS-Ca-Mg containing 15 mM glycine. After biotinylation, cell lysates were prepared as described above, and IRK was immunoprecipitated with an antibody directed against the {alpha}-subunit of the IRK. Immunoprecipitates were boiled in the presence of Laemmli buffer and subjected to SDS-PAGE. Proteins were transferred to Immobilon-P membranes and immunoblotted with {alpha} 960 or Streptavidin-HRP (Amersham Biosciences). Streptavidin binds to biotinylated proteins allowing only the detection of IRK associated with the PM.

Transferrin Uptake in Primary Hepatocytes—Transferrin (Tf) internalization in primary hepatocytes was performed as described previously (32). In brief, hepatocytes were preincubated in the presence or absence of 2 mM M{beta}CD for 1 h at 37 °C, and then 1 µgof 125I-Tf (around 2 µCi) was added. Iron-saturated Tf was labeled with 125I with the Chloramine-T method as described previously (33). After incubation at the indicated times at 37 °C, cells were washed three times in ice-cold PBS, incubated for 30 s with 250 mM acetic acid containing 500 mM NaCl followed by neutralization with 1 M NaAc, and washed again three times in ice-cold PBS. Cells were solubilized in 1% Triton X-100, and intracellular radioactivity was determined in a {gamma}-counter and normalized to the protein content. Specific incorporation of Tf into the hepatocytes was the difference between the binding 125I-Tf minus the binding 125I-Tf plus 400 µg of non-labeled iron saturated transferrin.

Glycogen Synthesis—Glycogen synthesis was determined by incorporation of [U-14C]glucose into glycogen as described previously (34). In brief, hepatocytes (1 x 106 cells) were serum-deprived for 4 h, pre-incubated in the absence or presence of 2 mM M{beta}CD for 1 h at 37 °C, and subsequently incubated for 2 h in serum-free media containing 15 mM [U-14C]glucose and insulin (100 nM), in the absence of M{beta}CD. Incubations were stopped by three rapid washes with ice-cold PBS, and cells were solubilized in 1 ml of 0.1 M NaOH. The samples were then boiled in the presence of 2 mg of carrier glycogen and precipitated overnight in 70% ethanol at –20 °C. After centrifugation, the precipitated glycogen was resuspended in 500 µl of water, incubated for 5 min at 70 °C, and incorporated radioactivity was determined by scintillation counting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Caveolin-1 Distribution in Rat Liver Subcellular Fractions— Caveolin-1 has been observed to be largely localized to the plasma membrane of selected cell types, in 50- to 100-nm {Omega}-shaped invaginations termed caveolae (10, 11). These caveolin-enriched microdomains are characterized by their light buoyant density and resistance to solubilization by Triton X-100 at 4 °C (10, 11). In this study, we examined the distribution of caveolin-1 in rat liver subcellular fractions by immunoblotting the various fractions using a specific antibody against this protein. As shown in Fig. 1a, caveolin-1 was detected principally in PM and microsomes and was barely detectable in endosomes or Golgi fractions.



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 1.
Distribution of caveolin-1 and actin in subcellular fractions from rat liver. Homogenate (H), microsomes (M), endosomes (EN), plasma membrane (PM), and Golgi (G) were purified from liver. Samples (50 µg of protein) were subjected to SDS-PAGE (6–12%), transferred to Immobilon-P membranes, and immunoblotted with antibodies specific for caveolin-1 (a) and total actin (b). WB, Western blotting.

 
Isolation of CMD from Rat Liver PM with Triton X-100 —We subsequently analyzed the distribution of caveolin-1 in rat liver PM after incubation of PM preparations with 1% Triton X-100 and subsequent sucrose gradient flotation analysis as described under "Experimental Procedures." Caveolin-1 was found in the Triton X-100 insoluble pellet and at the 10–30% sucrose interface (Fig. 2a, left, fractions 4 and 5), which constituted DRM with a buoyant density characteristic of lipid rafts (DRM/rafts). When corrected for protein content in each fraction, approximately 60% of caveolin-1 was found in the pellet, 10% in DRM/rafts at the 10–30% sucrose interface, and the remainder was solubilized as indicated by its presence in the load zone (fraction 9) beneath the sucrose gradient (Fig. 3d, white bars). Caveolin-1 was enriched in the pellet and the DRM/rafts (10–30% sucrose interface) by 11- and 7-fold, respectively, relative to the original PM lysate.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 2.
Isolation of CMDs and DRMs/rafts from PM and microsomes. a, plasma membranes and microsomes were prepared as described under "Experimental Procedures." These cell fractions were then homogenized in presence of Triton X-100 (final concentration, 1%) and mixed with an equal volume of 80% sucrose (final concentration, 40%). Each preparation (4 ml) was placed at the bottom of a centrifuge tube and was overlaid with 4 ml of 30% and 4 ml of 10% sucrose and centrifuged at 29,000 x g for 21 h. One-milliliter samples were collected from the top (fractions 1 to 8) and proteins were concentrated by precipitation with TCA-deoxycholate. The 40% sucrose layer (fraction 9) and the pellet (re-suspended in 1 ml of PBS) were also collected. Each of the eight concentrated fractions, and fraction 9 (50 µg of protein/fraction) as well as the re-suspended pellet were subjected to SDSPAGE (6–12%) and analyzed by immunoblotting with an anti-caveolin-1 (a) and anti-actin antibodies (b). A representative Western blot from one of three different experiments is shown. c, SDS-PAGE (6–12%) and silver staining of sucrose gradient fractions prepared from microsomes and PM (10 µg of protein). WB, Western blot.

 



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 3.
Characterization of CMDs in rat liver PM. Rats were fasted overnight and liver PM was prepared as described under "Experimental Procedures." PM preparations were re-suspended in Tris-buffered saline, pH 7.5, containing protease inhibitors and divided into three equal aliquots. These aliquots were homogenized in presence of 1% Triton X-100 (a), Na2CO3, pH 11 (b), and the combination of 1% Triton X-100 and Na2 CO3, pH 11 (c). These homogenates were placed at the bottom of a centrifuge tube and processed as described in Fig. 2. Fractions 1–8 and fraction 9 correspond to the 10–30% sucrose gradient and the soluble proteins of the 40% sucrose layer, respectively. The pellet was re-suspended in 1 ml of PBS. Each of the 9 fractions and the re-suspended pellet were subjected to SDS-PAGE (6–12%) and analyzed by immunoblotting with anti-caveolin-1 and anti-actin antibodies. A representative immunoblot of the three different solubilization conditions is depicted. The volume of each fraction and the volume loaded for SDS-PAGE are shown at the bottom of c. Bands were quantified by scanning densitometry and normalized to the total amount of caveolin-1 and actin in each fraction. The amount of caveolin-1 (d) and actin (e) are expressed as a percentage of the total amount in the original PM lysate (% total). Each point is the mean ± S.E. of three independent experiments. The protein content in each fraction is shown in f. WB, Western blotting.

 
The PM fractions we prepared have been characterized in detail and seem to be quite pure, although there is some contamination with endoplasmic reticulum (35). Microsomes contain both caveolin-1 (Figs. 1a and 2a, right) and abundant endoplasmic reticulum elements. Therefore, it is possible that a substantial portion of PM caveolin-1 is a result of contamination with microsomes. We therefore subjected microsomes treated with Triton X-100 to sucrose gradient fractionation and compared caveolin-1 distribution from this compartment (Fig. 2a, right) with that from PM. Caveolin-1 from microsomes was found in DRM/rafts and in the soluble fraction (fraction 9). In contrast to PM, no caveolin-1 was found in the Triton-insoluble pellet from microsomes (Fig. 2a, right), indicating a different distribution of caveolin-1 in these two compartments. To further compare the DRM/rafts from PM with those from microsomes, we subjected them to SDS-PAGE and subsequent silver staining. As seen in Fig. 2c, the DRM/rafts from PM displayed a protein pattern distinctly different from that observed in those isolated from microsomes, indicating their distinction from one another.

Caveolin-1 and the PM Cytoskeleton—It has been noted previously that the PM preparation used in our work contains abundant filaments (35, 36). Furthermore, after treatment of the PM with Triton X-100 and subsequent centrifugation, the filamentous structures in the resultant pellet were found to be enriched in actin (36). In our study, we found that actin was enriched only in the PM fraction, and not microsomes, Golgi, or endosomes, relative to its concentration in whole liver homogenate (Fig. 1b, compare lanes H and PM). When the distribution of actin was analyzed in PM subfractions, resolved by sucrose density centrifugation, we found that, as with caveolin-1 (Fig. 2b, left), actin was enriched in the pellet and the DRM/rafts (10–30% sucrose interface) by 5- and 2-fold, respectively, relative to the original PM lysate. We also found in microsomes that actin, in parallel with caveolin-1 distribution, was found in DRM/rafts and the soluble proteins of fraction 9, whereas none was found in the pellet (Fig. 2b, right). These results suggest that the PM microdomains containing caveolin-1 also contain insoluble actin-rich structures, perhaps in association with one another.

Characterization of CMD in Rat Liver PM—CMD have also been prepared by treating membranes with Na2CO3, pH 11 (37). It was shown previously that treatment of rat liver PM preparations with Na2CO3, pH 11, solubilized the filaments associated with the membrane but left the latter intact (36). Because a high proportion of caveolin-1 in PM is associated with the Triton X-100 insoluble pellet (Fig. 2a, left), we carried out sucrose gradient flotation analysis of Na2CO3-treated PM to dismantle the cytoskeleton and thus determine the characteristics of this pool of caveolin-1. PM lysates were incubated with Na2CO3, pH 11, and/or Triton X-100 and subsequently fractionated by sucrose density centrifugation (Fig. 3). When PM lysates were treated only with Na2CO3, pH 11, a substantial proportion was found at the interface between 10 and 30% sucrose (Fig. 3, b, top, and d, black bars) with some observed in fractions 6 to 8. As observed previously (36) after Na2CO3 treatment, actin was also removed from the pellet (Fig. 3b, bottom) and now largely appeared as soluble components (fraction 9) but also in association with entities of a buoyant density comparable with CMDs (Fig. 3, compare a and b, bottom; see also Fig. 3e, black bars).

To evaluate further the characteristics of the CMD associated with the Triton-insoluble pellet, PM were treated with a combination of Triton X-100 and Na2CO3, pH 11, before sucrose gradient fractionation. This treatment released most of the actin and caveolin-1 from the pellet (Fig. 3, c–e, gray bars). Thus, we found that the bulk of caveolin-1 and actin were now present as soluble entities in fraction 9 (Fig. 3, c–e, black bars). In addition, around 10% of caveolin-1 was present in the sucrose gradient but in less dense fractions compared with those derived from treating PM with Triton X-100 alone (Fig. 3, compare a and c, top). Because prior treatment of PM with both, Triton X-100 and Na2CO3, pH 11, removed actin (Fig. 3, c, bottom, and e) and other peripheral proteins (e.g. IRS1 and p85, data not shown) from the DRM/rafts fraction, we suggest that the CMD contained in this fraction have reduced density because of the removal of associated peripheral proteins. The total protein content in each of these fractions is shown in Fig. 3f.

In summary, the treatment of PM with a combination of Triton X-100 and Na2CO3, pH 11, demonstrates that only 10% of the PM pool of caveolin-1 is found in DRM/rafts, whereas the remainder is in CMD found in fraction 9 and presumably solubilized (Fig. 3d, gray columns). Around 60% of this latter Triton-soluble pool of caveolin-1 is tightly associated with the cytoskeleton (Fig. 3d, white bars) and is released from the cytoskeleton as low buoyant density CMD in the presence of Na2CO3 alone (Fig. 3d, black bars). The remaining 35%, which appears in fraction 9 after treatment with either Triton (Fig. 3d, white bar) or Na2CO3 (Fig. 3d, black bar), seems to represent a distinct population of CMD that has not yet been characterized.

CMD in Cultured Primary Rat Hepatocytes—To confirm the presence of CMD in parenchymal cells, we then examined primary rat hepatocyte cultures. Rat liver hepatocytes were homogenized in 1% Triton X-100 followed by sucrose gradient flotation analysis as described under "Experimental Procedures." As shown in Fig. 5a (black circles and control Western blot), the distribution of caveolin-1 in rat liver hepatocytes was similar to that observed in liver PM (compare with Fig. 2a, left), suggesting that the main source of caveolin-1 in liver are the parenchymal cells. CMD and rafts are rich in cholesterol, and partial extraction of the latter from cells is a commonly used method to disrupt the morphology and function of caveolar structures (38). We sought to use M{beta}CD to selectively extract cholesterol from the hepatocyte cell surface (39) and thus characterize CMDs therein. However, M{beta}CD can also remove cholesterol from PM domains other than CMD/rafts (38). For example, it has been observed that 10 mM M{beta}CD strongly reduced both transferrin and epidermal growth factor internalization, indicating a perturbation of clathrin-dependent internalization (40). We therefore initially sought to determine the concentration of M{beta}CD, which selectively affects CMD at the cell surface without disturbing the rest of the PM.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 5.
Effect of 2 mM M{beta}CD on CMD in hepatocytes. Primary hepatocytes were preincubated in presence ({circ}) or absence () of 2 mM M{beta}CD for 1 h at 37 °C, followed by homogenization with 1% Triton X-100 and sucrose gradient flotation analysis as described under "Experimental Procedures." The amount of protein loaded onto the gradient was normalized within each experiment. Fractions were subjected to SDS-PAGE (6–12%) and analyzed by immunoblotting with an anticaveolin-1 (Cav-1) antibody. The level of caveolin-1 was quantified by scanning densitometry and normalized to the total amount of caveolin-1 in each fraction. a, the amount of caveolin-1 is expressed as a percentage of the total amount in the original PM lysate (% of total). A representative immunoblot is shown at the bottom of the bar graph. Each point is the mean of three independent experiments. b, the content of Caveolin-1 in M{beta}CD treated cells is expressed as a percentage of control hepatocytes. Each bar is the mean ± S.E. of three independent experiments. *, p < 0.05.

 
To do this we employed a method that evaluates the integrity of the PM based on the accessibility of sulfo-biotin, used to label cell surface proteins (31), to the cellular interior. Therefore, we determined the maximal concentration of M{beta}CD that minimally effected permeability by incubating primary hepatocytes with different concentrations of M{beta}CD (0.5–10 mM), followed by labeling the cells with sulfo-biotin. Cell lysates were then prepared and immunoprecipitated with an antibody against the intracellular protein p85 (the regulatory subunit of PI3-kinase). Immunoisolated p85 was subjected to SDS-PAGE, transferred to Immobilon-P membranes, and analyzed by blotting with streptavidin, which binds with high affinity to biotin. As seen in Fig. 4a, in control hepatocytes and cells treated with 0.5, 1.0, and 2 mM M{beta}CD, little or no p85 was labeled with biotin. However, at higher concentrations of M{beta}CD, the reaction of sulfo-biotin with p85 was readily observed and increased progressively. These results indicate that a concentration of M{beta}CD higher than 2 mM disrupts the integrity of PM.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of M{beta}CD on the integrity of the hepatocyte PM and clathrin-dependent internalization. a, primary hepatocytes were incubated with the indicated concentrations of M{beta}CD for 1 h at 37 °C. Cell surface proteins were biotinylated as described under "Experimental Procedures." Hepatocytes were then lysed and PI3-kinase was immunoprecipitated from these lysates using an antibody against to p85. Immunoprecipitates were boiled in Laemmli sample buffer and subjected to SDS-PAGE. The gel proteins were transferred to Immobilon-P membranes and probed with streptavidin-HRP for detection of the pool of p85 labeled with biotin. b, uptake of 125I-transferrin (Tf) into hepatocytes: primary hepatocytes were preincubated in the presence or absence of 2 mM M{beta}CD for 1 h at 37 °C, and 125I-Tf or 125I-Tf plus unlabeled Tf were then added and hepatocytes were further incubated for 15 or 60 min. The level of intracellular transferrin was determined as indicated under "Experimental Procedures." The results are expressed as the mean ± S.E. of three separate experiments. c, IRK internalization: primary hepatocytes were incubated with or without 2 mM M{beta}CD for 1 h at 37 °C followed by addition of insulin (100 nM) for the indicted times. Cell surface proteins were biotinylated and lysed as indicated under "Experimental Procedures." IRK was immunoprecipitated from these lysates using an antibody to the {alpha} subunit of IRK. Immunoprecipitates were processed as indicated above and Immobilon-P membranes were probed with an anti-IRK antibody ({alpha} 960) or with streptavidin-horseradish peroxidase for detection of the total amount of IRK or PM-associated IRK, respectively. The depicted experiment is one of two similar ones. WB, Western blot.

 
To confirm the lack of any effect of 2 mM M{beta}CD on non-CMD domains, we investigated the effect of 2 mM M{beta}CD on the internalization of transferrin and IRK. Transferrin uptake into hepatocytes, pre-treated with 2 mM M{beta}CD, was measured after incubation with transferrin for 15 or 60 min. As seen in Fig. 4b, 2 mM M{beta}CD had no effect on the internalization of transferrin. We used cell surface biotinylation to asses the effect of M{beta}CD on insulin-induced IRK internalization. Hepatocytes were pre-incubated in presence or absence of 2 mM M{beta}CD followed by stimulation with insulin for the indicated times. Cell surface proteins were subsequently labeled with Sulfo-biotin, lysates were prepared, and IRKs immunoprecipitated. The levels of both total and PM-associated IRK were detected by immunoblotting with {alpha} 960 and streptavidin respectively. It can be seen in Fig. 4c that 2 mM M{beta}CD did not inhibit insulin-induced IRK internalization.

Having demonstrated that 2 mM M{beta}CD has a minimal effect on both the integrity of the PM and clathrin-dependent internalization, we then determined the effect of 2 mM M{beta}CD on CMDs in hepatocytes. As shown in Fig. 5, a and b, M{beta}CD induced the redistribution of caveolin-1 from the Triton X-100 insoluble pellet to the fraction 9 (Fig. 5, a and b). In Triton-treated hepatocytes, the distribution of caveolin-1 at the 10%/30% sucrose gradient interface (fractions 4 and 5) reflects CMDs coming from both PM and microsomes. However, as shown in Fig. 2a, all the caveolin-1 found in the Triton X-100 insoluble pellet originates from the PM. Therefore, treatment of hepatocytes with 2 mM M{beta}CD produces a dissociation of caveolin-1 from PM-associated cytoskeletal elements to the soluble fraction. Although 2 mM M{beta}CD could also affect caveolin-1 in DRM/rafts derived from PM, this effect would be masked by the presence of caveolin-1 DRM/rafts from microsomes, which are not accessed by 2 mM M{beta}CD. In summary, these observations confirm the presence of caveolin-1 in hepatocytes and also demonstrate that M{beta}CD can selectively affect CMD at the PM of the hepatocytes.

Insulin-induced Redistribution of Caveolin-1 and Actin in PM Subcellular Fractions—As shown in Fig. 6, the distribution of caveolin-1 was profoundly affected after treatment of the rats with insulin (1.5 µg/100 g of body weight). The caveolin-1 content in the Triton-insoluble pellet and in the total PM increased by 30 s and substantially subsided by 5 min after insulin treatment. In contrast, the amount of caveolin-1 in DRM/rafts dramatically decreased in the same interval of time (Fig. 6a). The insulin-stimulated increase of caveolin-1 content in total PM may reflect the translocation of caveolin-1 to PM from other cellular compartments. In the same time interval (zero to 5 min) after insulin, the amount of actin in total PM was not altered (Fig. 6b, black symbols). However, the actin content of the Triton-insoluble pellet increased significantly at 30 s and 5 min after insulin by 122 ± 8% and 146 ± 13%, respectively (Fig. 6b, white symbols); and decreased in the DRM/rafts isolated at the 10–30% sucrose interface (Fig. 6b, black triangles). We suggest that this reflects the promotion by insulin of actin reorganization in hepatocytes.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6.
Insulin-stimulated caveolin-1 and actin redistribution in PM subcompartments. After an overnight fast, rats received a single i.v. dose of insulin (1.5 µg/100 g of body weight) and were killed at the noted times thereafter. Liver PM were prepared and homogenized with 1% Triton X-100 as described under "Experimental Procedures." The amount of protein loaded onto the gradient was normalized within each experiment, and the preparations were processed as described in Fig. 2. Lipid raft ({blacktriangleup}), pellet ({circ}), and total PM () fractions were subjected to SDS-PAGE (6–12%) and analyzed by immunoblotting with anti-caveolin-1 (a) or total actin (b) antibodies. The level of caveolin-1 and actin in each fraction was quantified using scanning densitometry. The results were plotted as a percentage of the value in basal animals (no insulin treatment). Each point is the mean ± S.E. of three to seven independent experiments. *, p < 0.01; #, p < 0.05 (versus basal, by Student's t test).

 
Impact of Insulin on the Distribution of Caveolin-1 in PM Subcellular Fractions—We considered several possibilities to explain the disappearance of caveolin-1 from PM DRM/rafts after insulin stimulation. Previous reports have described an interaction between the insulin receptor and caveolin-1 (15, 16). Thus, a complex formed by IRK and caveolin-1 could be internalized into endosomes. We therefore evaluated the content of caveolin-1 in endosomes after insulin stimulation. As observed previously (41), insulin induced a rapid increase of IRK in endosomes at 2 min with a return to near basal levels by 15 min (Fig. 7a). In the same time interval, no caveolin-1 was detected in endosomes (Fig. 7a). Thus, internalization of caveolin-1 to endosomes is unlikely to account for the insulin-dependent decrease of caveolin-1 associated with DRM/rafts as noted in Fig. 6a.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 7.
Insulin stimulates the association of CMD in DRM/rafts with the PM cytoskeleton. After an overnight fast, rats received a single i.v. dose of insulin (1.5 µg/100 g of body weight) and were killed at the noted times thereafter. a, endosomes were prepared as described under "Experimental Procedures." Samples (50 µg of protein) were subjected to SDS-PAGE (6–12%), transferred to polyvinylidene difluoride membranes, and blotted with antibodies against IRK or caveolin-1 (Cav-1). A representative immunoblot of two independent experiments is shown. b, PM preparations, derived from insulin-treated rats (5 min), were split into two equal aliquots. One aliquot was incubated with 1% Triton X-100 and the other with a combination of 1% Triton X-100 and Na2CO3, pH 11, as described in Fig. 3. PM fractions were isolated as described in Fig. 2, subjected to SDS-PAGE (6–12%), and analyzed by immunoblotting with anti-caveolin-1 and actin antibodies. A representative immunoblot of three independent experiments is shown. WB, Western blot.

 
To determine whether CMD contained in DRM/rafts associate with the Triton-insoluble pellet after insulin stimulation, we examined the combined effect of Na2CO3, pH 11, and Triton X-100 on the distribution of caveolin-1 as seen in Fig. 3c. Thus, the PM preparation from insulin-stimulated (5 min) rats was divided into two aliquots; one half was homogenized in the presence of 1% Triton X-100 and the other half was homogenized with 1% Triton X-100 and Na2CO3, pH 11, before subfractionation by sucrose gradient centrifugation. When PM was isolated from rats receiving insulin and treated with Triton X-100 alone, we observed low levels of caveolin-1 in buoyant elements compared with PM from control rats (Figs. 6a and 7b, top). However, when PM from insulin-treated rats was solubilized in Triton X-100 and Na2CO3, pH 11, we observed the appearance of caveolin-1 in structures with a range of buoyant density (Fig. 7b, bottom, fractions 1 to 5). We suggest that this indicates insulin-induced association of the CMD contained in the DRM/rafts fraction with the Triton-insoluble actin-cytoskeleton of PM and that treatment with Na2CO3, pH 11, dissociates these CMD from their association with cytoskeletal elements. We suggest that their greater buoyancy reflects the removal by Na2CO3 of associated peripheral proteins as discussed in respect to Fig. 3c.

Distribution of IRK, IRS1, and p85 in Triton-insoluble Compartments of PM—In view of a number of studies indicating that insulin signaling in adipocytes involves caveolin-containing structures (12, 23) and our observations above, we investigated the distribution of IRK and downstream signaling molecules in PM subcompartments after insulin treatment (Fig. 8). More than 90% of the insulin receptor content of PM derived from control rats was solubilized with 1% Triton X-100. Only a small amount (around 2%) was found in DRM/rafts, and almost nothing was detectable in the pellet (Fig. 8a, 0 min). After insulin treatment, IRK increased transiently in DRM/rafts and progressively in the pellet. In both fractions, the IRK was tyrosine phosphorylated (Fig. 8a, 0.5–5 min). Previous work showed that Triton X-100 can solubilize IRK located in CMD (14). Because we found low levels of IRK in the Triton-insoluble compartments derived from PM, we determined the content of IRK in CMD isolated in the absence of Triton X-100 using magnetic beads coated with anti-caveolin-1 antibodies to selectively prepare CMD structures. With this technique, the amount of IRK detected in CMD was more abundant than that seen in DRM/rafts (Fig. 8b). Thus, it seems that a substantial proportion of IRK is localized to CMD in liver and is solubilized by treatment with 1% Triton X-100, as observed previously (14).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 8.
Effect of Insulin on IRK, p85, and IRS1 Distribution in PM Subfractions. After an overnight fast rats received a single i.v. dose of insulin (1.5 µg/100 g of body weight) and were killed at the noted times thereafter. Liver PM subcompartments were isolated as indicated in Fig. 2, subjected to SDS-PAGE (6–12%), and analyzed by immunoblotting with an anti-IRK or anti-phosphotyrosine (PY) antibodies (a). A representative immunoblot of three independent experiments is shown. b, immunoisolation of caveolin-enriched membranes: PM preparations were homogenized in Na2CO3, pH 11. Homogenates were centrifuged; the pellets were re-suspended in Tris-buffered saline, pH 7.5, and incubated with magnetic beads coated with an antibody specific for caveolin-1 or IgG (negative control) as described under "Experimental Procedures." Immunoisolated caveolin-1-containing structures were subjected to SDS-PAGE (6–12%) and analyzed by immunoblotting with anti-caveolin-1 and anti-IRK antibodies. A representative immunoblot of two independent experiments is shown. c, lipid rafts ({blacktriangleup}), pellets ({circ}), and total PM () fractions were subjected to SDS-PAGE (6–12%) and analyzed by immunoblotting with anti-IRS1 or p85 antibodies. The levels of these proteins were quantified using scanning densitometry. Top, representative immunoblot. Bottom, levels of IRS1 and p85 were plotted as a percentage of the value in non-insulin treated (basal) animals. Each point is the mean ± S.E. of three to seven independent experiments (*, p < 0.01; #, p < 0.05 [versus basal, by Student's t test]). WB, Western blot.

 
After sucrose density fractionation of PM homogenized in 1% Triton X-100 from control rats, approximately 5–8% of p85 and IRS1 were found in DRM/rafts (fractions 4 and 5) and 20–30% in the pellet (Fig. 8c). As is evident on comparing Fig. 8c with Fig. 2, a and b, IRS1 and p85 show substantial co-localization with caveolin-1 and actin. In a previous study, we showed that after insulin treatment, p85 was recruited to liver PM (6). In the current study, we have found that the bulk of p85 translocated to PM after insulin was directed to the Triton-insoluble pellet (Fig. 8c).

Effect of Cholesterol Extraction on Insulin Signaling in Primary Hepatocytes—The insulin-dependent translocation of CMD with the characteristics of DRM/rafts, IRK, and several important signaling molecules to the PM cytoskeleton suggests that these events are coupled and relevant for insulin signaling. To assess this hypothesis further, we examined insulin signaling in rat primary hepatocytes after disrupting CMD using M{beta}CD. We used concentrations of M{beta}CD up to 2 mM, which, as shown in Figs. 4 and 5, selectively disrupt PM-CMD. Pre-treatment of hepatocytes with 1 or 2 mM M{beta}CD had no effect on insulin-induced IRK tyrosine-phosphorylation (Fig. 9a). However, insulin-induced Akt phosphorylation/activation was decreased when hepatocytes were pre-incubated with 1 or 2 mM M{beta}CD (Fig. 9, b and c). Such an inhibition was more pronounced at 15 min than at 2 min after insulin stimulation (Fig. 9, b and c). The antibody used in this study to assess Akt activation detects mainly Akt1 phosphorylated at Ser-473. Because recent studies suggest that Akt2, but not Akt1, is involved in the metabolic actions of insulin (42), we have also used a specific antibody against Akt2 that detects both the phosphorylated and non-phosphorylated Akt2. As shown in Fig. 9d, M{beta}CD inhibits insulin-induced Akt2 activation. Similar to Akt1, Akt2 inhibition was particularly noticeable at 15 min after insulin. We also assessed the impact of cholesterol depletion on glycogen synthesis, which is dependent in hepatocytes on PI3-kinase/Akt activation (34). As seen in Fig. 9e, 2 mM M{beta}CD significantly inhibited the effect of insulin on glycogen synthesis. Taken together, our data suggest that there is a correlation between the association of CMDs with the cytoskeleton and insulin signaling.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 9.
Effect of M{beta}CD on insulin-induced IRK tyrosine-phosphorylation, Akt-Ser473 phosphorylation, and glycogen synthesis. Hepatocytes were incubated with or without M{beta}CD (1 or 2 mM) for 1hat37 °C followed by insulin (100 nM) for the indicated times. Lysates were prepared and equal amounts of protein (50 µg) were subjected to SDS-PAGE and transferred to Immobilon-P membranes. Membranes were immunoblotted with {alpha} 960 and {alpha} phosphotyrosine (PY) (a), {alpha} Akt and {alpha} Akt-Ser473 (b), and a specific antibody against Akt2 (d). The immunoblots shown in a and b are representative of two independent experiments with similar result. Results in c are the mean ± one half of the range in two independent experiments. Control (), 1 mM M{beta}CD ({circ}), 2 mM M{beta}CD ({blacktriangleup}). d, two independent experiments are shown. e, serum-starved hepatocytes were preincubated in the absence or presence of M{beta}CD (1 or 2 mM); subsequently, M{beta}CD was removed from the media and cells were further incubated with 15 mM [U-14C]glucose in the presence or absence of insulin (100 nM) for 2 h at 37 °C. Glycogen synthesis was determined as described under "Experimental Procedures." The results are expressed as the means ± S.E. of an experiment performed in triplicate (*, p < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Liver contains a low level of caveolin-1 compared with other tissues, especially adipocytes (43). Although it was suggested that the source of caveolin-1 in liver could be endothelial and not parenchymal cells (44), several studies have shown clearly the presence of caveolin-1 in hepatocytes with negligible amounts in rat liver endothelial cells (24, 25, 45). In addition, the presence of caveolin has been demonstrated in caveolae at the surface of hepatocytes (25). In our work, we too have demonstrated the presence of caveolin-1 in primary rat hepatocytes. We showed a similar distribution of caveolin-1 in liver and hepatocytes after Triton X-100 solubilization and sucrose flotation analysis (compare Figs. 2a and 5), further supporting the view that parenchymal cells are a significant source of caveolin-1 in liver. Thus, our data are consistent with the previous work of others (24, 25).

At the surface of the adipocytes or endothelial cells, caveolin-1 is found in invaginated structures termed caveolae, which are characterized by their light buoyant density and resistance to solubilization in Triton X-100 (10). However, our results show that most caveolin-1 at the PM is found in light buoyant structures, which are soluble in Triton X-100 and are associated with the PM cytoskeleton (Fig. 3). Only 10% of caveolin-1 was found in DRMs/rafts. Detergent-soluble caveolin have been identified in intracellular compartments (46, 47). Because our data on PM are not the result of contamination by internal membranes (see Fig. 2), we conclude that at least two populations of CMD exist in PM of the hepatocytes that must differ in their lipid and protein composition (Fig. 3). The insolubility of rafts/caveolae in Triton X-100 is determined, at least in part, by the high level of cholesterol and lipid with saturated acyl chains associated with these domains. Our data showing that around 90% of caveolin-1 at the PM, located in lipid domains soluble in cold Triton X-100 (Fig. 3), suggest that this pool of caveolin-1 may not be enriched in cholesterol. However, a low concentration of M{beta}CD (2 mM) partially disrupted the pool of Triton-soluble caveolin-1 associated with the cytoskeleton without affecting the integrity of the PM or clathrin-mediated internalization (Fig. 4). Thus, caveolin-1 would seem to be in lipid domains, enriched in cholesterol but nevertheless solubilized in cold Triton X-100. Consistent with this hypothesis, previous work has shown that PM derived from hepatocytes possess caveolin-1 organized in caveolae together with structures containing scattered caveolin (25). In addition, it has been proposed that there is a coexistence in the apical PM of different cholesterol-enriched lipid rafts based on their relative solubility in non-ionic detergents, such as Triton X-100 or Lubrol WX (48).

Insulin Signaling: A Role for CMD in PM—In response to insulin, we found that CMD associated with the DRM/rafts fraction of the PM rapidly disappeared (Fig. 6a). This was not caused by internalization of caveolin-IRK complexes into endosomes (Fig. 7a). Indeed, in response to insulin, these buoyant density domains become associated with PM-cytoskeletal elements (Fig. 7b). Besides CMD of DRM/rafts, insulin also induces rapid recruitment of IRK, p85, and IRS1 to a cytoskeleton-enriched fraction derived from PM (Fig. 8). All these events were accompanied by rapid actin reorganization as evidenced in Fig. 6b, suggesting that the actin cytoskeleton could be the target of CMD and signaling proteins. This hypothesis is supported by previous studies carried out in L6 myotubes, which demonstrated that, in response to insulin, p85 was translocated to newly formed actin-containing structures (49). PI3-kinase and subsequent Rac-1 activation may be involved in this effect because both wortmannin (49–52) and a dominant-negative Rac-1 (49) inhibited insulin-stimulated actin reorganization and signaling. A close relation between caveolae/raft and the actin cytoskeleton has been previously observed. Thus the F-actin cross-linking protein filamin associates with caveolin-1, and both proteins co-localize with stress fibers when analyzed by immunofluorescence (53). In addition, it was recently found that 3T3 L1 adipocytes have a unique cortical F-actin structure that is associated with raft/caveolae at the PM (54). In summary, our results suggest that 1) insulin induces the association of CMD with the characteristics of caveolae and insulin-signaling molecules with the PM actin-cytoskeleton and 2) CMD seem to be relevant for insulin signaling in hepatocytes. We have also compared the effect of epidermal growth factor on the distribution of caveolin-1 and signaling molecules in CMD isolated from PM with those of insulin. Epidermal growth factor induced rapid recruitment of p85, actin, and caveolin-1 to the raft fraction with no increase of these proteins in the cytoskeleton-enriched fraction.2 Thus, the insulin effects observed in this study seem to be specific for this hormone.

The presence of IRK in CMD of DRM/rafts could be considered a strong argument in favor of the significance of caveolae for insulin signaling. However, the available data concerning this issue are not clear. This work, as well as an earlier study, show that IRK is barely detectable in CMD isolated with Triton X-100 (17). In contrast, we observed substantial levels of IRK in PM-CMD prepared by immunoisolation with an anti-caveolin-1 antibody attached to magnetic beads in absence of detergent (Fig. 8b). The presence of IRK in CMD has been confirmed by electron microscopy and immunofluorescence in 3T3 L1 adipocytes (14). In this work, the authors showed that the IRK co-fractionated with CMD when Na3CO2, pH 11, but not Triton X-100 was used to extract CMD. They suggested that this was caused by selective solubilization of IRK by this detergent (14). Our data conform to this observation. In contrast to these results, Souto et al. (55) analyzed the presence of IRK in adipocyte caveolae by immunopurification of caveolae and electron microscopy combined with immunogold labeling. Both techniques showed that there is a lack of IRK in caveolae (55). In summary, we suggest that the presence of a functional IRK in PM-CMD together with an insulin-induced association of CMD of DRM/rafts with the cytoskeleton support the view that insulin signals through PM caveolae in liver.

Cholesterol Depletion in Primary Hepatocytes: Effect on Insulin Signaling—The constitutive association of Triton-soluble CMD with PM-cytoskeleton and the subsequent translocation of Triton-insoluble CMD (DRMs/rafts) to this latter compartment in response to insulin make almost impossible any further analysis of the role of CMD on insulin signaling in rat liver. Although treatment of the PM with Na2CO3, pH 11, can disrupt the association between the cytoskeleton and CMD, this also interferes with the association of these lipid domains with insulin-signaling proteins (i.e. IRS1, p85, etc.). Thus, to evaluate further the importance of CMD on insulin signaling in rat liver, we continued our studies in primary hepatocyte cultures. We used M{beta}CD to extract cholesterol from the PM of the hepatocytes as a tool to investigate the role of CMD on insulin signaling (38). CMD are enriched in cholesterol; therefore, these membrane domains are more sensitive to cholesterol extraction compared with the rest of the PM. However, the concentration of M{beta}CD used to extract cholesterol from CMD must be carefully determined because, as suggested in this and previous work (40), non-CMD PM is also affected by cholesterol depletion. Thus, in this work, we found that 2 mM M{beta}CD partially disrupted hepatocyte cell surface CMD without permeabilizing the cell to sulfo-biotin or affecting clathrin-dependent internalization (Fig. 4). Under this circumstance, extraction of cholesterol from hepatocyte PM did not interfere with the insulin-induced tyrosine-phosphorylation of IRK but inhibited the downstream activation of PKB/Akt. When a low concentration of M{beta}CD was used (1 or 2 mM), we observed partial inhibition of Akt activation, principally at 15 min but not at 2 min after insulin. Thus, our results suggest that CMD could be involved in the modulation of insulin signaling later after insulin administration. It is also of considerable interest that insulin-induced glycogen synthesis is inhibited by 2 mM M{beta}CD. This is in accordance with the dependence of insulin-induced glycogen synthesis on PI3-kinase/Akt activation in hepatocytes (34).

We have also demonstrated that clathrin-mediated endocytosis is not affected by 2 mM M{beta}CD because neither IRK nor transferrin internalization was inhibited when hepatocytes were preincubated with this concentration of M{beta}CD (Fig. 4). Thus, CMD could be essential for insulin signaling after IRK is internalized and may modulate the translocation of insulin-signaling proteins downstream of IRK to a specific subcellular compartment. In accordance with this hypothesis, a previous work carried out in adipocytes has shown that {beta}-cyclodextrin inhibits the insulin-induced association of IRS1 with IRK without affecting the activation of the latter (18). Thereafter, IRS1 tyrosine phosphorylation and Akt activation were inhibited (18). Although liver contains a low level of caveolin-1 compared with adipose tissue, cholesterol depletion in hepatocytes induces effects on insulin signaling similar to those observed in adipocytes. In agreement with a role of CMD in insulin signaling, it has been demonstrated recently that caveolin-1-null mice are insulin-resistant and also show a drastic reduction in the level of IRK in adipocytes (23). The author of this work hypothesized that caveolin-1 could be necessary for proper stabilization of IRK in adipocytes because IRK mRNA levels were not affected in caveolin-1-null mice (23). Our data are consistent with a role for CMD in insulin signaling in hepatocytes. Because this conclusion relies on the effect of M{beta}CD, which, as discussed above, is not absolutely specific for CMD, further work is required to define this role conclusively.

In summary, we report the existence of at least two different populations of CMD in rat liver PM. One population of CMD is soluble in Triton X-100 and seems to be constitutively associated with cytoskeletal elements. A second population of CMD is located in a membrane compartment insoluble in Triton X-100 with light buoyant density (DRM/rafts). These microdomains behave as dynamic structures in response to insulin. Thus, along with IRK, p85, and IRS1, CMD were found to associate, in an insulin-dependent manner, with actin-containing cytoskeletal structures. Finally, the negative effect of cholesterol depletion on insulin signaling supports the view that CMD participate in the regulation of insulin signaling in liver.


   FOOTNOTES
 
* This work was supported by the Canadian Institutes for Health Research and the National Cancer Institute of Canada. We appreciate the continuing generosity of the Cleghorn Fund at McGill University, and the Maurice Pollack Foundation of Montreal. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 514-398-4101; Fax: 514-398-3923; E-mail: barry.posner{at}staff.mcgill.ca.

1 The abbreviations used are: IRK, insulin receptor kinase; PI3-kinase, phosphatidylinositol 3-kinase; CMD, caveolin-1-enriched membrane domains; PM, plasma membranes; PBS, phosphate-buffered saline; DRM, detergent resistant membranes; M{beta}CD, methyl-{beta}-cyclodextrin; Tf, transferrin; IRS1, insulin receptor substrate 1; p85, regulatory subunit of PI3-kinase. Back

2 A. Balbis, G. Baquiran, and B. I. Posner, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Louise Larose, Simon Wing, and Amanda Parmar for the critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pignataro, O. P., and Ascoli, M. (1990) Mol. Endocrinol. 4, 758–765[Abstract]
  2. Carter, A. N., and Downes, C. P. (1992) J. Biol. Chem. 267, 14563–14567[Abstract/Free Full Text]
  3. Hu, P., Margolis, B., Skolnik, E. Y., Lammers, R., Ullrich, A., and Schlessinger, J. (1992) Mol. Cell. Biol. 12, 981–990[Abstract/Free Full Text]
  4. Di Guglielmo, G. M., Baass, P. C., Ou, W. J., Posner, B. I., and Bergeron, J. J. (1994) EMBO J. 13, 4269–4277[Medline] [Order article via Infotrieve]
  5. Bevan, A. P., Burgess, J. W., Drake, P. G., Shaver, A., Bergeron, J. J., and Posner, B. I. (1995) J. Biol. Chem. 270, 10784–10791[Abstract/Free Full Text]
  6. Balbis, A., Baquiran, G., Bergeron, J. J., and Posner, B. I. (2000) Endocrinology 141, 4041–4049[Abstract/Free Full Text]
  7. Baumann, C. A., and Saltiel, A. R. (2001) Bioessays 23, 215–222[CrossRef][Medline] [Order article via Infotrieve]
  8. Brown, D. A., and London, E. (1998) Annu. Rev. Cell Dev. Biol. 14, 111–136[CrossRef][Medline] [Order article via Infotrieve]
  9. Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 31–39[CrossRef][Medline] [Order article via Infotrieve]
  10. Anderson, R. G. (1998) Annu. Rev. Biochem. 67, 199–225[CrossRef][Medline] [Order article via Infotrieve]
  11. Kurzchalia, T. V., and Parton, R. G. (1999) Curr. Opin. Cell Biol. 11, 424–431[CrossRef][Medline] [Order article via Infotrieve]
  12. Bickel, P. E. (2002) Am. J. Physiol. 282, E1–E10
  13. Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998) J. Biol. Chem. 273, 5419–5422[Free Full Text]
  14. Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K. E., and Stralfors, P. (1999) FASEB J. 13, 1961–1971[Abstract/Free Full Text]
  15. Yamamoto, M., Toya, Y., Schwencke, C., Lisanti, M. P., Myers, M. G., Jr., and Ishikawa, Y. (1998) J. Biol. Chem. 273, 26962–26968[Abstract/Free Full Text]
  16. Nystrom, F. H., Chen, H., Cong, L. N., Li, Y., and Quon, M. J. (1999) Mol. Endocrinol. 13, 2013–2024[Abstract/Free Full Text]
  17. Mastick, C. C., Brady, M. J., and Saltiel, A. R. (1995) J. Cell Biol. 129, 1523–1531[Abstract/Free Full Text]
  18. Parpal, S., Karlsson, M., Thorn, H., and Stralfors, P. (2001) J. Biol. Chem. 276, 9670–9678[Abstract/Free Full Text]
  19. Baumann, C. A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E., and Saltiel, A. R. (2000) Nature 407, 202–207[CrossRef][Medline] [Order article via Infotrieve]
  20. Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E., and Saltiel, A. R. (2001) Nature 410, 944–948[CrossRef][Medline] [Order article via Infotrieve]
  21. Razani, B., and Lisanti, M. P. (2001) J. Clin. Investig. 108, 1553–1561