JBC Papers in Press. Published on July 12, 2004 as Manuscript M404280200 1 Effect of Insulin on Caveolin Enriched Membrane Domains in Rat Liver Alejandro Balbis, Gerardo Baquiran, Catherine Mounier, and Barry I. Posner Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, 3640 University St., D o Suite W315, Montreal, Quebec H3A 2B2, Canada w n lo a d ed fro m Running Title: Insulin signaling in liver: role of caveolin-enriched membranes http ://w w w .jb c .org b/ y gu e s t o n A p ril 1 0 , 2 0 1 9 To whom correspondence should be addressed: Dr Barry I Posner; Tel. (514) 398-4101; Fax: (514) 398-3923; E-mail: [email protected] Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc. 2 SUMMARY Compartmentalization of signaling molecules may explain, at least in part, how insulin or growth factors achieve specificity. Caveolae/rafts are specialized lipid compartments, which 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 appears 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. In response to insulin we found D o evidence of rapid actin reorganization in rat liver PM along with the association of CMD/rafts w n lo a d and insulin signaling molecules with a cell fraction enriched in cytoskeletal elements. The ed fro m presence of CMD in liver parenchyma cells was confirmed by the presence of caveolin-1 in http ://w primary rat hepatocyte cultures. Cholesterol depletion, effected by incubating hepatocytes with w w .jb c 2mM methyl - β - cyclodextrin (MßCD), did not permeabilize the cells or interfere with clathrin- .org b/ y g dependent internalization. However at this concentration MßCD perturbed CMD of hepatocyte u e s t o n PM, inhibited insulin-induced Akt activation and glycogen synthesis, but did not affect insulin- A p ril 1 induced IRK tyrosine phosphorylation. These events, together with the presence of a functional 0, 2 0 1 9 insulin receptor in CMD of rat liver PM, suggest that insulin signaling is influenced by the interaction of caveolae with cytoskeletal elements in liver. 3 INTRODUCTION Although binding of insulin to its receptor is a high affinity specific process, 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 question of how insulin achieves its specificity of action remains to be satisfactorily resolved. Compartmentalization of signaling molecules in plasma membranes and endosomes may play an important role in determining the specificity of signal transduction (4-7). D o Recently, the isolation of detergent-insoluble, low-density membrane fragments from cells w n lo a d suggests that sphingolipid and cholesterol rich domains could exist as a liquid-ordered phase in ed fro m the membrane (8). These lipid domains, which are known as lipid rafts, can recruit or exclude http ://w signaling proteins. Thus, lipid rafts have been implicated in the regulation of hormone and w w .jb c growth factor signaling (8,9). Caveolae, which constitute a subset of lipid rafts, are invaginated .org b/ y cell surface micro-domains, enriched in caveolin oligomers, the major protein constituent of gu e s t o n these structures (10,11). Three caveolins (caveolin 1, 2 and 3) have been discovered. Caveolin- A p ril 1 1 and caveolin-2 are found most abundantly in adipocyte and endothelial cells, whereas 0 , 2 0 1 9 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 EGF 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 ß-cyclodextrin, attenuated IRK signaling (14,18). Rafts/caveolae have also been implicated in insulin-stimulated glucose transport in 4 3T3-L1 adipocytes by a mechanism independent of PI3-kinase (19,20). Recently, caveolin-1 knockout mice have been created, which are viable despite a complete ablation of caveolae (21). These mice show impaired nitric oxide signaling, vascular dysfunction, hyper-proliferation 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 D o has nevertheless clearly been shown that caveolin-1 is located in liver parenchymal cells with w n lo a d negligible levels detected in endothelial cells (24,25). Furthermore, caveolae have been ed fro m demonstrated at the cell surface of hepatocytes using rapid-freeze deep-etching electron http ://w microscopy (25). Since liver is an important insulin target tissue we investigated the significance w w .jb c of caveolin enriched membrane domains (CMD) in mediating insulin signaling in rat liver. In the .org b/ y present study we have characterized these entities and have demonstrated a possible function gu e s t o n in insulin signaling. A p ril 1 0 , 2 0 1 9 5 EXPERIMENTAL PROCEDURES Animals- Female Sprague-Dawley rats, 10 weeks of age, (160-180g bodyweight (BW)) were purchased from Charles River 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, USA). Phenylmethanesulphonic fluoride (PMSF), sodium orthovanadate, methyl-ß-cyclodextrin and most other chemicals were purchased from Sigma (St Louis, MO, USA). Reagents for electrophoresis were from Bio-Rad (Richmond, CA, USA). Kodak X-OMAT AR film was from D o Picker International (Montreal, PQ, Canada). PVDF Immobilon-P transfer membranes were from w n lo a d Millipore Ltd. (Mississauga, ON, Canada). [U-14C]glucose (300 mCi / mmol) was purchased from ed fro m NEN Life Sciences Products-DuPOnt (Wilmington, DE). Magnetic beads (Dynabeads M-280) http ://w were from Dynal (Lake Success, NY). An antibody raised against a peptide corresponding to w w .jb c residues 942-969 of the juxtamembrane region of the IRK ß-subunit (anti-960) was prepared .org b/ y and purified on a PAS column as previously described (26 ) and used for Western blotting. For gu e s t o n immunoprecipitation of IRK, an antibody directed against the α-subunit was obtained from the A p ril 1 serum of a patient with acanthosis nigricans (27). Polyclonal anti-p85, a polyclonal anti-IRS1 0, 2 0 1 9 antibody and a specific antibody against Akt2 were purchased from Upstate Biotechnology Incorporated (Lake Placid, NY, USA). Antibodies against Akt1 and phospho-Akt1 (Ser473) were purchased from New England Biolabs, Inc. (Mississauga, Canada). An antibody against actin and iron saturated transferrin were purchased from Sigma (St Louis, MO, USA). A monoclonal anti-phosphotyrosine antibody (anti-PY) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caveolin-1 was purchased from Transduction Laboratories (Lexington, KY). Preparation of Sub-cellular Fractions- Rats were anaesthetized and sacrificed by decapitation at the indicated times following intra-jugular injections as described in the 6 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 PMSF, 1 mM MgCl , 2mM NaF, and 2 mM Na VO ). Plasma membranes 2 3 4 (PM), endosomes (ENs), and Microsomes were prepared as previously described (27). A purified Golgi fraction prepared as described (28) was kindly provided by Dr. Bergeron. The protein content of these fractions was measured using a modification of Bradford’s method with BSA as standard (29). Isolation of Caveolin Enriched Membrane Domains (CMD) – 1) Isolation of CMD with Triton X-100: CMD were isolated by a modification of the method of Liu et al (30). Briefly, plasma D o membranes were pelleted and mixed with 3 ml of ice-cold 1% Triton-X100 in buffer A (50 mM w n lo a d Tris-HCl [pH 7.5], 150 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF and 2 mM ed fro m Na3V04). The samples were homogenized (10 strokes in glass homogenizer), incubated on ice http ://w for 1 hr, adjusted to the same amount of protein, and diluted 1:1 with 80 % sucrose in buffer B w w .jb c (50 mM Tris-HCl [pH 7.5] - 150 mM NaCl). The extract (4 ml, between 2 and 5 mg of protein) .org b/ y was loaded on the bottom of a 12 ml ultracentrifuge tube and overlaid with 4 ml each of 30% gu e s t o n and 10% sucrose in buffer B. The gradient was centrifuged for 21 h at 29,000 x g in a SW40 Ti A p ril 1 rotor (Beckman Instruments) and 1 ml fractions were collected from the top of the tube. 0 , 2 0 1 9 Fractions 1-8 (10-30% sucrose gradient), fraction 9 (soluble proteins in the residual 40% sucrose layer), and the pellet, re-suspended in 1ml of ice cold phosphate buffered saline (PBS) were subsequently analyzed by SDS-PAGE and Western Blotting. The amount of protein recovered in the DRM/lipid raft fraction (fractions 4 and 5 of sucrose gradient) and in the Triton insoluble pellet was between 20-100 µg and 400-700 µg respectively. 2) Isolation of CMD with a detergent free method: Briefly, PM was mixed with 3 ml of ice-cold Na CO (200 mM, pH 11) in 2 3 buffer A. Samples were homogenized (10 strokes in glass homogenizer), sonicated (3 times, 10 seconds each) and incubated on ice for 1 hr. Subsequently, samples were mixed with 80% sucrose, centrifuged overnight and collected as indicated above. 3) In some experiments PM 7 were homogenized in 1% Triton X-100 and Na CO (pH 11; 200 mM final concentration) in 2 3 buffer A. After incubating on ice for 1 hr 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 µl of 3x 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 re-suspended in 1 ml of PBS buffer, mixed with 500 µl of 3 x Laemmli sample buffer and aliquots of 100 µl were used for SDS-PAGE. Fifty µg of protein D o from fraction 9 and total PM respectively were used for SDS-PAGE. Immunoblotting with anti w n lo a d caveolin-1 antibody showed a non-specific band at 29 kDa when total membranes or fraction 9 ed fro m were analyzed. This band was absent in the Triton-insoluble pellet and in lipid rafts. Either http ://w I125GAR or I125GAM were used as secondary antibody and, following autoradiography at -80°C, w w .jb c appropriate bands were quantified using a BioRad GS-700 Imaging Densitometer. .org b/ y Immuno-isolation of CMDs - PM was homogenized in Na2CO3 (pH 11) (10 strokes Dounce gue s t o n Homogenizer) and incubated on ice for 1 h followed by centrifugation at 200,000 x g for 1 h. A p ril 1 The resultant pellet was again homogenized and now sonicated (3 times, 10 seconds each) in 0 , 2 0 1 9 Tris buffered saline (TBS, buffer B) containing proteases inhibitors and 1% albumin as noted above. The treatment with Na CO was carried out in order to remove PM filaments, which could 2 3 interfere with the immuno-isolation 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 TBS (pH 7.5) prior to boiling for 5 minutes 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., St. Constant, Canada) by in situ liver perfusion with collagenase were 8 plated on a collagen matrix (Vitrogen-100). Sub-confluent cultures were prepared by seeding 1 x 106 cells, onto 9.6 cm2 six-well plates (Corning, Costar, Cambridge, MA) or 5 x 106 cells, onto 78 cm2 culture dishes (Starstedt Canada, St. Laurent, Canada). Cells were bathed for 24 h in seeding medium (DMEM/Ham’s F-12 containing 10% FBS, 10 mM HEPES, 20 mM NaHCO , 3 500 IU/ml penicillin, and 500 µg/ml streptomycin) and then for 48 h in serum-free medium (SFM) 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 MßCD. Biotinylation of Cell Surface Proteins - Detection of IRK located at the cell surface of the D o hepatocytes was performed as described previously (31). Briefly, hepatocytes were incubated in w n lo a d the presence or absence of MßCD and insulin as noted in Figure Legends. Thereafter, ed fro m hepatocytes were washed three times with ice-cold PBS-Ca-Mg pH: 7.4 (0.1mM CaCl2 and 1 http ://w mM MgCl ) and cell surface proteins were biotinylated by incubation with 0.5 mg/ml of Sulfo- w 2 w .jb c NHS-LC-Biotin (Pierce, Rockford, IL) in PBS-Ca-Mg for 30 min at 4°C. The reaction was .org b/ y stopped by washing the dishes three times with PBS-Ca-Mg containing 15 mM of glycine. After gu e s t o n biotinylation, cell lysates were prepared as described above and IRK was immunoprecipitated A p ril 1 with an antibody directed against the α-subunit of the IRK. Immunoprecipitates were boiled in 0, 2 0 1 9 the presence of Laemmli buffer and subjected to SDS-PAGE. Proteins were transferred to Immobilon-P membranes and immunoblotted with α960 or Streptavidin-HRP (Amersham Pharmacia Biotech, Piscataway, USA). 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). Briefly, hepatocytes were pre-incubated in the presence or absence of 2 mM MßCD for 1 hr at 37°C, and then 1µg of 125I-Tf (around 2 µCi) was added. Iron- saturated Tf was labeled with 125I with the Chloramine-T method as previously described (33). After 9 incubation at the indicated times at 37°C, cells were washed three times in ice-cold PBS, incubated for 30 seconds with 250 mM of acetic acid containing 500 mM of NaCl followed by neutralization with 1 M of 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 previously described (34). Briefly, hepatocytes (1×106 cells) were serum- deprived for 4 h, pre-incubated in absence or presence of 2 mM of MßCD for 1 hour at 37 °C D o and subsequently incubated for 2 hours in serum free media containing 15 mM [U-14C]glucose w n lo a d and insulin (100 nM), in absence of MßCD. Incubations were stopped by three rapid washes ed fro m with ice cold PBS and cells were solubilized in 1 ml of 0.1 M NaOH. The samples were then http ://w boiled in the presence of 2 mg of carrier glycogen and precipitated over-night in 70% ethanol at w w .jb c -20°C. After centrifugation, the precipitated glycogen was resuspended in 500 µl of water, .org b/ y g incubated for 5 min at 70°C, and incorporated radioactivity was determined by scintillation u e s t o n counting. A p ril 1 0 , 2 0 1 9 10 RESULTS 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 micro-domains (CMD) 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 Figure 1a, caveolin-1 was detected principally in PM and microsomes, and was barely detectable in endosomes or Golgi fractions. D o wn lo a d e Isolation of CMD from Rat Liver PM with Triton X-100 d fro m We subsequently analyzed the distribution of caveolin-1 in rat liver PM following incubation of http ://w w PM preparations with 1% Triton X-100 and subsequent sucrose gradient flotation analysis as w .jb c .o described in Experimental Procedures. Caveolin-1 was found in the Triton X-100 insoluble pellet rg b/ y g and at the 10-30% sucrose interface (Fractions 4 & 5 in Fig.2a, left panel), which constituted u e s t o n detergent resistant membranes (DRM) with a buoyant density characteristic of lipid rafts A p ril 1 (DRM/rafts). When corrected for protein content in each fraction, around 60% of caveolin-1 was 0, 2 0 1 9 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) to the extent of 11 and 7 fold respectively relative to the original PM lysate. The PM fractions we prepared have been characterized in detail and appear to be quite pure although there is some contamination with endoplasmic reticulum (ER) (35). Microsomes contain both caveolin-1 (Figs. 1a and 2a, right panel) and abundant ER elements. Therefore it is possible that a substantial portion of PM caveolin-1 is due to contamination with microsomes.