(Received for publication, May 2, 1995; and in revised form, January 31, 1996)
From the
Caveolin is believed to play an important role in sorting processes, vesicular trafficking, transmembrane signaling, and molecular transport across membranes. In this study we have evaluated the expression and distribution of caveolin in skeletal muscle and its interaction with GLUT4 glucose carriers. Caveolin was expressed to substantial levels in muscle and its expression was regulated in muscle; aging and high fat diet enhanced caveolin expression in skeletal muscle and inversely, myogenesis down-regulated caveolin in L6E9 cells.
Under fasting conditions, most of caveolin was found in intracellular membranes and the caveolin present in the cell surface was found in both sarcolemma and T-tubules. Insulin administration led to a redistribution of caveolin from intracellular high density membrane fractions to intracellular lighter density fractions and to the cell surface; this pattern of insulin-induced redistribution was different to what was shown by GLUT4. These results suggests that caveolin is a component of an insulin-regulated machinery of vesicular transport in muscle.
Quantitative immunoisolation of GLUT4 vesicles obtained from different intracellular GLUT4 populations revealed the absence of caveolin which substantiates the lack of colocalization of intracellular GLUT4 and caveolin. This indicates that caveolin is not involved in intracellular GLUT4 trafficking in skeletal muscle.
Insulin stimulates glucose transport in skeletal muscle by a
process that is characterized by an enhancement of V values(1, 2, 3, 4) . In
parallel, insulin causes the recruitment of GLUT4, the main glucose
carrier expressed in skeletal muscle, from an intracellular compartment
to selective domains of sarcolemma and to
T-tubules(5, 6, 7, 8, 9, 10, 11) .
Insulin is not the only effector that causes redistribution of GLUT4 in
skeletal muscle; acute exercise has been demonstrated to cause
recruitment of GLUT4 to the cell
surface(12, 13, 14) . There is not yet direct
evidence on whether exercise recruits GLUT4 to similar cell surface
domains and by translocation of similar intracellular GLUT4
compartments in skeletal muscle(12, 13, 14) .
Important for the understanding of GLUT4 translocation is the knowledge of the proteins that colocalize with GLUT4 in the same compartment. A lack of colocalization of GLUT4 and TGN38 protein both in 3T3 adipocytes (15) and in rat skeletal muscle (11) has been substantiated, which indicates that the trans Golgi network is not a major site of the intracellular GLUT4 pool in adipocytes and in the muscle fiber. Recent studies have identified some of the proteins that colocalize in GLUT4-containing vesicles from rat adipocytes and skeletal muscle. Among the proteins that colocalize with GLUT4 in intracellular vesicles, the presence of phosphatidylinositol 4-kinase(16) , VAMPs (17) , SCAMPs/GTV3(18, 19) , gp160(20, 21, 22) , and some low molecular GTP-binding proteins including rab4 (23, 24) have been reported.
Caveolin is a principal component of the coat component of caveolae (25) and a major phosphoprotein in v-Src-transformed cells(26) . Caveolin is believed to play an important role in sorting processes, transmembrane signaling and molecular transport across membranes(27) . The relevance of caveolin to GLUT4 traffic has been recently investigated in 3T3-L1 and rat adipocytes(28, 29) . Scherer et al.(28) have suggested that caveolae may play an important role in the vesicular transport of GLUT4 in 3T3-L1 adipocytes. In contrast, Kandror et al.(29) have concluded that caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in isolated rat adipocytes. Here, we have studied the expression and distribution of caveolin in skeletal muscle, the regulation by insulin of caveolin distribution, and the possible role of caveolin on GLUT4 traffic.
In some assays, antibodies 1F8 (5-7 µg) and 3F8 (3 µl) were incubated overnight at 4 °C with goat anti-mouse IgG- or goat anti-mouse IgM-coupled to agarose (75 µl of bead volume). Beads were collected by a 6-s spin in a Microfuge and washed in PBS. Intact membrane preparations (15-25 µg of protein) were incubated with 1F8- or 3F8-Ig-agarose overnight at 4 °C in the absence of detergents (0.1% bovine serum albumin, 1 mM EDTA in PBS; final volume, 200 µl). The agarose beads and vesicles bound to them were collected by a 6-s spin in a Microfuge. The vesicles that were bound to the immobilized antibody were washed in PBS. The adsorbed material was eluted with electrophoresis sample buffer.
Figure 1: Expression of caveolin and GLUT4 in myoblast and myotube L6E9 cells. Total membranes were obtained from myoblast and myotube L6E9 cells. The abundance of caveolin and GLUT4 was determined by immunoblot analysis by using specific antibodies (see ``Experimental Procedures''). Representative autoradiograms from two to seven experiments are shown.
In another set of experiments, we analyzed the muscle content of caveolin and GLUT4 in two different experimental conditions known to alter GLUT4 expression in skeletal muscle, i.e. aging and high fat feeding(50, 51, 52) . To this end, we compared the expression of caveolin and GLUT4 in skeletal muscle from 3- and 12-month-old rats. In addition, we also studied the expression of proteins of 12-month-old rats subjected to a 6-month period of high fat feeding. The level of GLUT4 decreased 39% in muscle from aged rats compared to values found in the control group (Fig. 2). Furthermore, levels of GLUT4 further decreased (46% decrease) in muscle from aged rats as a result of high fat feeding (Fig. 2). In contrast, caveolin levels in skeletal muscle increased by 74% in aged rats compared to young rats (Fig. 2) and a high fat diet further increased the levels (99% increase compared to aged rats subjected to regular diet) (Fig. 2).
Figure 2: Effect of aging and high fat diet on caveolin and GLUT4 expression in skeletal muscle. Total membrane proteins were purified from skeletal muscle obtained from 3-month-old rats (Y) and 12-month-old rats either subjected to regular diet (Ag) or to a high fat diet (H). The abundance of caveolin and GLUT4 was determined by immunoblot analysis by using specific antibodies (see ``Experimental Procedures''). Representative autoradiograms from seven to 10 separate experiments are shown.
Figure 3:
Caveolin and GLUT4 are mainly found in
intracellular membranes in skeletal muscle. The abundance of caveolin,
GLUT4, -integrin, dihydropyridine receptor and
sarcoplasmic Ca
-ATPase in cell surface is shown as
the percentage of each specific protein found in cell surface membranes (A) and the results are mean ± S.E. of four to six
separate experiments. The abundance of caveolin, GLUT4, and
-integrin was assayed in surface membrane fractions SM1, SM2, and TT (B) and
in intracellular membranes 26F1, 29F1, 35F1, 26F2, 29F2, and 35F2 (C) from rat
skeletal muscle. The distribution of the caveolin, GLUT4,
-integrin, dihydropyridine receptor, and sarcoplasmic
Ca
-ATPase was determined by immunoblot analysis by
using specific antibodies (see ``Experimental Procedures'').
Equal amounts of membrane proteins (1 µg for GLU4 and
-integrin and 4 µg for caveolin) from the
different fractions were laid on gels. Representative autoradiograms
from four to seven experiments are shown in B and C.
The autoradiograms presented in B and C were exposed
for different time periods.
We have recently reported that
agglutination of cell surface membrane fractions from skeletal muscle
leads to the isolation of three distinct cell surface domains:
sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) a T-tubule
fraction(37) . In order to determine the localization of
caveolin, comparatively to GLUT4, in the different cell surface
membranes obtained from skeletal muscle, Western blotting of these
proteins was performed on fractions SM1, SM2 and T-tubules. In keeping
with previous observations(37) , fractions SM1 and SM2 showed a
high abundance of -integrin, whereas the T-tubule
fraction showed a low abundance of
-integrin (Fig. 3B). In keeping with previous observations, GLUT4
was present in all cell surface fractions, i.e. SM1, SM2, and
T-tubules, and GLUT4 abundance was significantly greater in the
T-tubule than in fractions SM1 or SM2 (Fig. 3B).
Similar to the pattern of GLUT4 distribution, caveolin was found in
substantial levels in all cell surface membranes analyzed (Fig. 3B). Caveolin and GLUT4 also showed a similar
pattern of distribution in the different intracellular membranes
obtained (Fig. 3C). Synaptophysin, a protein found in
peripheral nerve tissue (53) was not detected in cell surface
or in intracellular membranes (data not shown).
Figure 4: Insulin redistributes caveolin and GLUT4 in membrane fractions from skeletal muscle. The abundance of caveolin and GLUT4 was assayed in cell surface membrane fractions 23F1 and 23F2 and in intracellular membranes 26F1, 29F1, 35F1, 26F2, 29F2, and 35F2 from control and insulin-stimulated muscles. The distribution of caveolin and GLUT4 was determined by immunoblot analysis by using specific antibodies. Equal amounts of membrane proteins (1 µg for GLUT4 and 4 µg for caveolin) from the different fractions were laid on gels. Representative autoradiograms, obtained after various times of exposure, from two to seven separate experiments are shown.
The abundance of caveolin was increased in response to insulin in fractions 23F1 (a cell surface membrane population) and 26F1 (an intracellular membrane fraction) (levels of caveolin in the insulin-treated group increased by 73 and 72% compared to control values in 23F1 and 26F1, respectively). Under these conditions, the levels of caveolin were markedly decreased in the insulin-treated group in intracellular fractions 29F1 and 35F1 (levels of caveolin decreased in response to insulin by 95 and 94% in 29F1 and 35F1, respectively) (Fig. 4). No alterations in the abundance of caveolin were substantiated in fractions 23F2 (a cell surface membrane fraction) and 26F2, 29F2 and 35F2 (from intracellular origin) (Fig. 4). These data suggest that insulin redistributes caveolin in a complex manner from some intracellular high density membrane fractions to intracellular lighter density membranes as well as to the cell surface.
Figure 5: Subcellular localization of GLUT4 and caveolin in rat skeletal muscle. Rat skeletal muscle was homogenized in buffer containing 1% Triton X-100, brought to 40% sucrose and overlaid with a linear 5-30% sucrose gradient lacking detergent. After centrifugation for 12-16 h at 39,000 rpm, 12 fractions were harvested. An equal volume of each fraction was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting for the distribution of caveolin and GLUT4.
We have identified two distinct pools of intracellular GLUT4-containing membranes in skeletal muscle based on their differing insulin response and whereas one pool (membrane fractions 26F2, 29F2, and 35F2) was insulin-insensitive, the other one (membrane fractions 26F1, 29F1, and 35F1) showed a marked decrease in GLUT4 content after insulin administration (Fig. 4). In order to determine whether intracellular caveolin and GLUT4 colocalize in the muscle fiber, vesicle immunoisolation assays were performed using antibody 1F8 (against GLUT4) coupled to acrylic beads, using insulin-sensitive (i.e. fraction 26F1) or insulin-insensitive (i.e. fraction 26F2) intracellular membrane fractions. Antibody 1F8 immunoadsorbed nearly 90% and 89% of total GLUT4 from the fractions 26F1 and 26F2, respectively (Fig. 6, A and B). Under these conditions, antibody 1F8 did not immunoadsorb caveolin (Fig. 6, A and B). We also analyzed whether some degree of colocalization between caveolin and GLUT4 was detected in insulin-sensitive GLUT4 pools after in vivo insulin treatment. To this end, immunoadsorption assays were performed by incubating fractions 26F1 and 29F1 (insulin-sensitive intracellular membrane fractions) from control and insulin-treated muscles with immobilized antibody 1F8. Antibody 1F8 immunoadsorbed smaller amounts of GLUT4 in the insulin-treated group than in control when starting with fraction 26F1 (data not shown). Under these conditions, caveolin was absent from these vesicle population (data not shown). Identical results were obtained with fraction 29F1 (data not shown).
Figure 6: Absence of caveolin in intracellular insulin-sensitive and insulin-insensitive GLUT4 pools. Membrane vesicles 26F1 (insulin-sensitive GLUT4 pool) (A) and membrane vesicles 26F2 (insulin-insensitive GLUT4 pool) (B) obtained from from nonstimulated skeletal muscle were incubated with (+) or without(-) antibody 1F8. After the incubation, the adsorbed (P) and nonadsorbed (S) fractions were electrophoresed and immunoblotted to determine the abundance of caveolin and GLUT4. Autoradiographs were subjected to scanning densitometry. Representative autoradiograms, obtained after various times of exposure, are shown.
In other studies, monoclonal antibody 3F8 directed against SCAMPs was also used in vesicle immunoisolation of insulin-sensitive and insulin-insensitive GLUT4 pools. Antibody 3F8 immunoadsorbed nearly 38 and 47% of total SCAMP 37 and 18 and 31% of total GLUT4 from fractions 26F1 and 26F2, respectively (data not shown). However, under these conditions, no caveolin was specifically immunoadsorbed (data not shown).
The results of this study demonstrate an abundant expression of caveolin in muscle and its regulation by myogenesis, aging and high fat feeding. We have also found that most of caveolin is distributed intracellularly in skeletal muscle under basal conditions and that acute insulin administration redistributes caveolin from intracellular high density membranes to the cell surface and to intracellular lighter density membranes. These results suggest that caveolin participates in vesicular traffic in skeletal muscle in an insulin-regulatable manner. However, intracellular caveolin does not colocalize with SCAMP proteins, markers of the general cell surface recycling system(54, 55) , or with the GLUT4 glucose transporter isoform. In consequence, the vesicular traffic machinery in which caveolin participates is different than the system associated with SCAMPs and, at least in skeletal muscle, it is also unrelated to the vesicular traffic of GLUT4.
Our data emphasize the importance of understanding the biological role of caveolin in skeletal muscle. This is based on two findings, namely that caveolin is abundantly expressed in skeletal muscle and that its expression is subjected to regulation. That muscle is an abundant source of caveolin is substantiated by the fact that the expression of caveolin in this tissue, when expressed per gram of tissue, accounts for 10% of values detected in adipose tissue, which is the most abundant source of caveolin(28) . We have also demonstrated that aging and high fat feeding, two situations characterized by muscle insulin resistance (56, 57, 58) caused an enhancement of caveolin levels in skeletal muscle. The biological impact of the up-regulation of caveolin with aging or high fat feeding is unknown and the possible implication of caveolin up-regulation and muscle insulin resistance remains undetermined.
We have also observed that the differentiation of L6E9 cells into myotubes is associated with decreased cellular levels of caveolin. This does not necessarily mean a decrease in abundance of caveolar organelles during differentiation. In this regard, it has very recently reported a novel homologue of caveolin termed M-caveolin, which is expressed exclusively in skeletal muscle and heart and its expression is induced upon muscle differentiation in C2C12 cells(59) . The mechanisms by which caveolin is abundantly expressed in the adult rat skeletal muscle but not in cultured myotubes deserves further study.
Caveolin is an integral part of the striated coat of caveolae, i.e. non-clathrin-coated invaginations of the plasma membrane. Caveolin seems to internalize from caveolae to intracellular compartments in response to okadaic acid(60) . Cholesterol oxidation also causes the movement of caveolin from caveolae to the Golgi apparatus (61) in the absence of changes in the number or morphology of caveolae. This together with the observation that caveolin is an integral component of trans-Golgi network-derived transport vesicles (62) supports the view that caveolin is a component of the molecular machinery of vesicular transport. In this regard, we have found that in skeletal muscle, caveolin is mainly distributed intracellularly. Under basal conditions, 77% of all caveolin found in muscle membranes was detected intracellularly. This distribution pattern is similar to the distribution of SCAMP proteins (63) and to GLUT4 glucose transporter in skeletal muscle. The distribution of caveolin in intracellular membranes from rat skeletal muscle suggests a functional role in vesicular transport.
Under our experimental conditions, we found that in vivo insulin administration caused a marked redistribution of caveolin in skeletal muscle. Thus, caveolin moved from high density intracellular membranes to the cell surface and to intracellular membranes of lower density. This is in keeping with previous observations in 3T3-L1 adipocytes indicating that insulin increased the amount of caveolin present in the plasma membrane and a decrease in the amount of caveolin associated with low density microsomes(28) . This pattern of insulin-induced redistribution of caveolin is fairly similar to the effect of insulin on the distribution of SCAMPs in skeletal muscle (63) . However, the redistribution of caveolin was markedly different than the insulin-induced translocation of GLUT4. The biological role of the insulin-triggered redistribution of caveolin in skeletal muscle deserves further study.
It is important to determine whether caveolin and GLUT4 interact in the muscle fiber. This aspect has been studied in this report in a number of ways. Caveolin-rich membrane domains were isolated from rat skeletal muscle which revealed that less than 10% of total GLUT4 overlapped with caveolin-containing fractions. Additionally, the colocalization of GLUT4 and caveolin in intracellular membranes was done by vesicle immunoadsorption analysis. In these studies we used two distinct intracellular GLUT4 pools previously obtained from rat skeletal muscle(63) : one intracellular GLUT4 pool that is insulin-sensitive since insulin administration causes a large depletion of this pool, and a second pool that is insulin-insensitive in terms of GLUT4 content. Vesicle immunoisolation assays clearly indicated that caveolin was absent from these distinct intracellular GLUT4-containing membranes obtained from muscle under basal or insulin-stimulated conditions. This allows us to conclude that caveolin is not an intermediate in intracellular trafficking of GLUT4 in skeletal muscle. If the overlapping found between GLUT4 and caveolin-rich membrane domains was interpreted as real colocalization between GLUT4 and caveolin, these data together with results obtained from vesicle immunoisolation analysis would be consistent with the idea that caveolin-rich membrane domains act as transient intermediates in GLUT4 endocytosis and/or exocytosis in muscle. This hypothesis requires extensive experimental work.
In any case, our studies indicate that caveolin is not an intermediate in intracellular trafficking of GLUT4 in skeletal muscle, as found in isolated rat adipocytes (29) and in contrast to what has been suggested in 3T3-L1 adipocytes(28) . The reason for this discrepancy is at present unknown but it should be mentioned that 3T3-L1 adipocytes also shows marked differences compared with isolated rat adipocytes in the trafficking of GLUT1 and GLUT4, and whereas GLUT1 and GLUT4 largely colocalize in 3T3-L1 they are completely seggregated in rat adipocytes (64, 65, 66) .
We also searched for colocalization of caveolin with SCAMPs proteins. SCAMPs are expressed in rat skeletal muscle where they show an intracellular distribution(63) . Insulin has been found to alter the cellular distribution of SCAMPs in adipocytes and in skeletal muscle and a variable extent of colocalization has been reported between intracellular SCAMPs and GLUT4 in adipocytes(18, 19, 63) . To determine whether SCAMPs and caveolin colocalize in skeletal muscle membranes, intracellular SCAMP-containing vesicles were immunoisolated with immobilized antibody 3F8. Under conditions, in which we immunoisolated near 40-50% of SCAMPs, caveolin was absent from the immunoprecipitates. In consequence, we conclude that there is a seggregation of SCAMPs and caveolin in intracellular membranes from rat skeletal muscle. In summary, based on the pattern of cellular distribution caused by insulin and on the lack of colocalization between caveolin and GLUT4 or SCAMPs, we propose the activity of, at least, three separate insulin-regulated mechanisms for vesicular transport in skeletal muscle. One system is characterized by the presence of caveolin, the second is characterized by the presence of SCAMPs and the third is involved in the recycling of GLUT4 glucose carriers.