1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx; 2Division of Hormone-dependent Tumor Biology, The Albert Einstein Cancer Center, Bronx; 3Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 7 January 2003 ; accepted in final form 19 March 2003
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ABSTRACT |
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caveolae; caveolin; insulin signaling; protein stabilization; knockout mice
In 1992, an explosion in the field occurred with the discovery of caveolin (now called caveolin-1) as a protein marker and principal component of caveolae (32). This allowed for the development of purification techniques that permitted the biochemical study of caveolae microdomains.
In subsequent years, two other caveolins were discovered, now termed caveolin-2 and caveolin-3 (37, 44). The tissue distributions of the various caveolins have been well studied, and it is now widely accepted that caveolin-1 is found most abundantly in the adipocyte, endothelial, and epithelial cells, whereas caveolin-3 is found in skeletal and cardiac myocytes. The distribution of caveolin-2 most closely follows that of caveolin-1, where it forms functional heterooligomers with caveolin-1 of about 1416 individual subunits (36, 44).
Caveolae are now known to be much more than the endocytic vesicles they were once thought to be. Many new discoveries have led to the proposal of the "caveolae signaling hypothesis," which attributes the regulation of numerous signaling molecules to a direct relationship with the caveolins (22). Regarding caveolin-1, the majority of these relationships are those of inhibition, as is the case with molecules such as endothelial nitric oxide synthase, Src family tyrosine kinases, EGF receptor, G proteins, and PKA (30, 45). Recently, however, caveolin-1 has also been shown to activate certain signaling molecules, i.e., the insulin receptor (21, 45).
With the development of caveolin-1-deficient (Cav-1 null) mice, a new area of research has emerged, because all of the proposed functions of caveolin-1 can now be tested in vivo. The first publications regarding this animal (6, 27, 28, 31) describe a viable and fertile mouse with a hyperproliferative lung phenotype, exercise intolerance, and, more recently, abnormalities in lipid homeostasis.
In our report on this subject (31), we showed that Cav-1 null mice are resistant to diet-induced obesity and demonstrate significant white adipose tissue atrophy with age. Although the pathogenesis of these findings was not elucidated, we also showed that Cav-1 null mice are hypertriglyceridemic (in both the fasted and postprandial state) and have elevated serum levels of free fatty acids in the postprandial state. In addition, these mice are unable to clear a triglyceride load, as evidenced by the kinetic buildup of triglyceride-rich chylomicrons/very low-density lipoproteins in the blood after administration of a fat bolus. We also found that two adipokines, Acrp30 and leptin, a lack of which has been associated with insulin resistance, are significantly reduced in their plasma concentrations. These changes occurred without any noticeable differences in other plasma metabolites, such as glucose, insulin, or cholesterol.
Here, we elucidate the molecular mechanism underlying these adipocyte-based phenotypes in Cav-1 null mice. One of the major functions of insulin signaling in the adipocyte is to activate lipogenesis and inhibit lipolysis. Previous in vitro studies have shown that caveolin-1 can interact with the insulin receptor and enhance insulin-mediated phosphorylation of IRS-1 (21, 49). Thus, if these findings are indeed physiologically relevant, it would be predicted that loss of caveolin-1 should dampen insulin receptor signaling, leading to unregulated lipolysis and a reduction of lipogenic signals. In support of this notion, we show that Cav-1 null mice develop postprandial hyperinsulinemia on a high-fat diet and demonstrate insulin insensitivity. Furthermore, we show that these defects are due to a primary defect in insulin signaling in the adipocyte at the level of the insulin receptor itself.
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MATERIAL AND METHODS |
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Animal studies. All animals were housed and maintained in a barrier facility at the Institute for Animal Studies, Albert Einstein College of Medicine. Cav-1 (/) null mice, Cav-2 (/) null mice, and the corresponding WT cohorts were generated and maintained by us, as previously described (28, 31).
High-fat diet study; insulin and glucose measurements. WT and Cav-1 null mice were placed at weaning (3 wk of age) on either a high-fat diet (59% of calories derived from fat; D12492 [GenBank] , Research Diets) or a normal chow diet (10% of calories derived from fat; D12450 [GenBank] B, Research Diets). At 9 mo, fasting and postprandial plasma insulin levels were measured by radioimmunoassay and fasting serum glucose levels were measured colorimetrically, as we previously described (27).
Insulin tolerance test (ITT). Weight-matched 20-wk-old mice that had been fed a normal chow diet were given an intraperitoneal injection of 0.75 U/kg insulin. Fasted serum glucose levels were then measured at all time points colorimetrically. It is important to note that WT and Cav-1 null mice do not display any statistically significant weight differences at this age (27), and thus the same dose of insulin was given to each mouse.
Muscle triglyceride analysis. Muscle triglycerides were extracted
according to the methods of Bligh and Dyer, as described by Iverson et al.
(11). Briefly, hindlimb muscle
tissue was removed from each mouse (WT, n = 7; Cav-1 null, n
= 7) and promptly frozen in liquid N2. About 400 mg of tissue were
homogenized in 4 ml of extraction buffer (20 mM Tris, pH 7.3, 1 mM EDTA, and 1
mM -mercaptoethanol). This extract (800 µl) was placed in a glass
tube, and 1 ml of chloroform and 2 ml of methanol were added, mixed, and
allowed to stand for 30 min. Next, 1 ml of water and 1 ml of chloroform were
added, and the samples were mixed and centrifuged for 20 min at 3,000 rpm at
10°C. The upper aqueous layer was discarded, and the lower phase was
evaporated under N2. The sample was redissolved in 250 µl of
isopropanol, and true triglycerides were measured using GPO-Trinder (Sigma),
according to the manufacturer's instructions.
Immunofluorescence. Mouse embryo fibroblasts (MEFs) were grown on
glass coverslips, fixed in 2% paraformaldehyde, and permeabilized in 0.1%
Triton X-100. Cells were then immunostained with monoclonal anti-caveolin-1
(cl 2234) and polyclonal anti-insulin receptor- antibodies. Bound
primary antibodies were visualized with a fluorescein-conjugated anti-mouse
antibody and a rhodamine-conjugated anti-rabbit antibody (Jackson
ImmunoResearch). As expected, omission of the primary antibodies prevented
immunostaining.
Immunoblot analysis. Mice were killed, and fat, liver, and muscle
samples were harvested and immediately frozen in liquid N2 (WT,
n 7; Cav-1 null, n
7). Approximately 100 mg of a
given tissue sample were then homogenized in lysis buffer [50 mM Tris, pH 8.0,
150 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 2 nM sodium orthovanadate,
0.1 µg/ml okadaic acid, 40 nM bpVphen, 1% Igepal (formerly NP-40), and 0.5%
deoxycholic acid] containing protease inhibitors (Boehringer Mannheim). Tissue
lysates were then centrifuged at 12,000 g for 10 min to remove
insoluble debris. Protein concentrations were analyzed using the BCA reagent
(Pierce), and the volume required for 40 µg of protein was determined.
Samples were then separated by SDS-PAGE and transferred to nitrocellulose. The
nitrocellulose membranes were stained with Ponceau S (to visualize protein
bands), followed by immunoblot analysis. All subsequent wash buffers contained
10 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween 20, which were supplemented
with 1% bovine serum albumin (BSA) and 2% nonfat dry milk (Carnation) for the
blocking solution and 1% BSA for the antibody diluent. Primary antibodies were
used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary
antibodies (1: 5,000 dilution; Pierce) were used to visualize bound primary
antibodies with the Supersignal chemiluminescence substrate (Pierce).
Northern blot analysis. Mice were killed, and the perigonadal fat pad was removed and frozen in liquid N2. Total RNA was extracted from 150 mg of tissue from each sample using the Trizol reagent protocol (GIBCO). Total RNA (20 µg) for each sample was separated by using a 1.2% agarose gel under RNase-free conditions and transferred to a Hybond-XL nylon membrane (Amersham). The filters were hybridized using the ExpressHyb solution (Clontech). An insulin receptor probe was generated by PCR amplification of a 1.5-kb fragment of the insulin receptor cDNA coding sequence by using custom-synthesized primers (5'-ATGAATTCCAGCAACTTGCTGTGC-3') corresponding to the first 24 bases and (5'-CTGGTTGCAAGCCTGCAGCTC-3') corresponding to 21 bases at the 1.5-kb point along the insulin receptor cDNA. This PCR product was then gel purified, radiolabeled, and hybridized to the nylon membrane.
Cell culture. Human embryonic kidney (HEK)-293 cells and MEFs (WT and Cav-1 knockout) were grown in DMEM supplemented with glutamine, antibiotics (penicillin and streptomycin), and 10% fetal calf serum.
Transient transfections. HEK-293 cells and Cav-1 knockout MEFs
were transiently transfected with the caveolin-1 cDNA (pCB7-Cav-1) or the
caveolin-1 61100 mutant [pCB7-Cav-1 (
61100)] using
either the standard calcium phosphate protocol (for HEK-293) or Lipofectamine
Plus reagent [for MEFs; according to the manufacturer's instructions
(Invitrogen)]. Thirty-six hours posttransfection, cells were collected into
lysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1% Triton X-100, and 60 mM octyl
glucoside) containing protease inhibitors (Boehringer Mannheim) and subjected
to either SDS-PAGE or Western analysis. Alternatively, cells were examined by
immunofluorescence microscopy.
Rescue of insulin receptor levels in Cav-1-deficient MEFs. MEFs were treated with either vehicle alone (Me2SO) or the proteasomal inhibitor MG-132 (1 µM; Sigma) for the time points indicated. Cells were then lysed and subjected to immunoblot analysis.
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RESULTS |
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We did not observe any insulin resistance in mice fed a normal diet, so we chose to mimic a factor that helps to produce this condition in the human population, namely, a high-fat diet. We therefore placed male and female cohorts of WT and knockout mice at weaning (3 wk of age) on either a high-fat diet (59% of calories from fat) or a normal chow diet (10% of calories from fat). At 9 mo of age, fasting and postprandial plasma insulin levels were measured by radioimmunoassay.
Figure 1 shows that all mice
exhibited the expected rise in insulin levels in the postprandial state.
However, the Cav-1 null mice that were fed a high-fat diet demonstrated
statistically significant postprandial hyperinsulinemia compared with WT mice
fed the same diet. Importantly, Cav-1 null mice that were fed a normal chow
diet did not show any hyperinsulinemia, suggesting that caveolin-1 plays a
role in diet-induced insulin resistance. In addition, no statistically
significant differences in fasting serum glucose concentrations were observed
between WT and Cav-1 null mice on a high-fat diet (females: WT, 126.7 ±
14.4 mg/dl; Cav-1 null, 129.8 ± 17.7 mg/dl; males: WT, 122.8 ±
7.1 mg/dl; Cav-1 null, 128.2 ± 11.0 mg/dl; n 6 mice for
each experimental group). It is important to note that all the other studies
detailed below were performed on young mice fed a normal chow diet.
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Cav-1 null mice show a blunted response to insulin, as assessed by ITT. Because hyperinsulinemia is generally a result of underlying insulin resistance, we next sought to determine whether Cav-1 knockout mice show any changes in insulin responsiveness. Weight-matched 20-wk-old Cav-1 null and WT mice that had been fed a normal chow diet were given an intraperitoneal injection of insulin (0.75 U/kg). Fasted serum glucose levels were then measured at all time points for all mice colorimetrically.
Figure 2 shows the expected decline in glucose levels after the administration of insulin (due to glucose uptake in peripheral tissues). Baseline glucose levels for both cohorts of mice are indistinguishable. However, most importantly, Cav-1 null mice showed a markedly blunted decline in glucose levels upon insulin injection, indicative of peripheral insulin resistance.
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Cav-1 null mice do not demonstrate fat deposition at ectopic sites. Obesity is associated with the development of insulin resistance and type II diabetes in humans and rodents. Although the cause of this association is not known, many have suggested that fat deposition outside of the adipocyte is to blame, the so-called "lipotoxicity theory" (8). This theory states that when lipid accumulates in insulin-sensitive tissues other than fat, an inhibition of insulin action results. Therefore, it is possible that ectopic fatty deposits are the cause of the observed postprandial hyperinsulinemia in Cav-1 null mice.
To this end, we used a modified Bligh and Dyer extraction protocol to measure the triglyceride content of hindlimb muscle in both WT and Cav-1 null mice. Muscle samples were removed from each mouse, weighed, and homogenized. A portion of this homogenate was then subjected to chloroform-methanol phase separation. After evaporation of the chloroform layer, total and free triglyceride contents were measured colorimetrically, from which the true triglyceride content was calculated. Our analysis shows that there is no significant difference between the lipid content of skeletal muscle obtained from Cav-1 null or WT mice (WT, 17.48 ± 4.14 µg/mg tissue; Cav-1 null, 17.84 ± 5.35 µg/mg tissue). Thus it appears that "lipotoxicity" does not play a role in the postprandial hyperinsulinemia observed in Cav-1 null mice.
Cav-1 null mice have a primary defect in insulin signaling in the adipocyte. Because it appears that insulin resistance in Cav-1 null mice is not due to ectopic fat deposits, we next turned our attention to the more intriguing culprit, the adipocyte. The observed postprandial hyperinsulinemia and insulin resistance are consistent with findings demonstrating a role for caveolin-1 in enhancing insulin signaling, so we sought to determine whether this phenotype is a result of a primary defect in the Cav-1 null adipocyte.
Cav-1 null and WT mice that had been fed a normal chow diet were
intraperitoneally injected with insulin (1 U/kg) for 7.5 min and then killed
by CO2 asphyxiation. Perigonadal fat pads were then immediately
removed and frozen in liquid N2. These samples were then
homogenized in lysis buffer, and equal amounts of protein were subjected to
Western blot analysis. Because the insulin receptor is composed of two
extracellular -subunits and two transmembrane
-subunits that are
normally tyrosine phosphorylated in response to insulin binding, we chose to
look at the phosphorylation state of the
-subunit as a marker for
insulin receptor activity. Interestingly, we found a dramatic reduction in
insulin receptor-
(IR-
) tyrosine phosphorylation/activation in the
Cav-1 knockout mouse (Fig.
3A). However, upon further analysis, we deduced that this
reduction in IR-
tyrosine phosphorylation is due to the downregulation
of the insulin receptor protein itself in Cav-1 null adipose tissue
(Fig. 3A). Thus Cav-1
null mice have an insulin receptor deficiency (>90% reduced) in adipose
tissue.
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To determine whether this reduction in IR- protein levels is
selective for adipose tissue, we next examined the status of IR-
levels
in other insulin-responsive tissues that do not contain an abundance of
caveolin-1, i.e., skeletal muscle and liver. As might be predicted, neither
liver nor skeletal muscle tissue samples from Cav-1 null mice showed any
changes in IR-
protein levels (Fig.
3, B and C). These findings provide the first in
vivo evidence for an interaction between caveolin-1 and the insulin receptor,
because it appears that caveolin-1 is necessary for insulin receptor
stabilization in adipocytes.
Cav-2 null mice do not show any changes in insulin receptor
levels. As we previously reported, caveolin-1 deficiency in mice leads to
an 95% reduction in caveolin-2 protein levels, because caveolin-1 protein
expression is required to stabilize the caveolin-2 protein product
(28,
29). Therefore, Cav-1 null
mice are essentially deficient in both caveolin-1 and caveolin-2.
To determine whether downregulation of insulin receptor protein levels in
Cav-1 null mice is due to the loss of caveolin-1 or caveolin-2, we also
examined insulin receptor levels in adipose tissue samples derived from Cav-2
null mice. As shown in Fig. 4,
the protein levels of insulin receptor- were not altered in Cav-2 null
mice. Therefore, we can conclude that the disruption of the insulin receptor
is dependent on the status of caveolin-1 but not caveolin-2. This is
consistent with our previous observations that Cav-2 null mice have normal
adipose tissue and clearly do not share the abnormal adipose tissue phenotype
observed in Cav-1 null mice
(31).
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Insulin receptor mRNA levels remain unchanged in Cav-1 null adipose tissue. To determine whether the change in insulin receptor protein expression is due to transcriptional downregulation in Cav-1 null mice, we next used Northern blot analysis to quantify the amount of insulin receptor mRNA in Cav-1 null adipose tissue. Total RNA was isolated from perigonadal fat pads using the Trizol reagent, separated on an agarose gel, and then transferred to a Hybond-XL nylon membrane. Primers were then used to construct a 1.5-kb probe by PCR from a plasmid containing the entire insulin receptor cDNA. The probe was gel purified, quantified, labeled with [32P]dCTP, and hybridized to the nylon membrane.
Our results show that there is no difference between the quantity of insulin receptor mRNA in Cav-1 null mice compared with WT mice. The 18S and 28S rRNA are shown as a control for equal loading (Fig. 5).
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Insulin-like growth factor-1 receptor protein levels remain unchanged
in Cav-1 null adipose tissue. The insulin receptor is one of a family of
receptor tyrosine kinases that includes the IGF-1 receptor (IGF-1R). Both
receptors are composed of structurally similar subunits, termed and
, that are known to form functional hybrid dimers in cells that have
both receptors. Furthermore, a certain degree of overlap is known to exist
between these two receptors for their native ligands, insulin and the IGFs
(20,
33).
Because the Cav-1 knockout mice are able to maintain euglycemia while the
level of insulin receptor protein is drastically decreased, we sought to
determine the role of the IGF-1R in glucose metabolism in these mice. As
described above, we removed perigonadal fat pads from killed mice, homogenized
them in lysis buffer, and subjected these lysates to SDS-PAGE. Immunoblotting
of nitrocellulose membranes with antibodies specific for IGF-1R showed
that there is no difference between WT and Cav-1 null mice in this regard
(Fig. 6). Thus there is no
compensatory upregulation of IGF-1R in Cav-1 knockout mice.
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GLUT-4 and PKB/Akt are upregulated in Cav-1 null adipose tissue. Because the signals generated by insulin and the insulin receptor itself are drastically diminished in Cav-1 null adipose tissue, we next examined the levels of several proteins downstream of the insulin receptor in the insulin signaling cascade to detect a possible compensatory upregulation of these signaling molecules.
Perigonadal fat pads were homogenized in lysis buffer and subjected to Western analysis. As shown in Fig. 7A, we found that the levels of GLUT-4 were dramatically increased in Cav-1 null mice, possibly as a response to the postprandial hyperinsulinemia. Other groups have previously reported that GLUT-4 levels are elevated in response to hyperinsulinemia (3, 5). Furthermore, a recent report indicates that, in 3T3L1 adipocytes, disruption of caveolae with cholesterol chelating agents results in an insulin-independent increase in plasma membrane GLUT-4 (39).
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We also found that PKB/Akt levels were increased
(Fig. 7B), perhaps in
response to reduced insulin signaling, because Akt normally functions to
induce GLUT-4 translocation to the membrane and promotes glycogen synthesis
via inactivation of GSK-3
(19,
46).
However, other proteins in the insulin signaling cascade remained unchanged
in their expression levels (Fig. 7,
CE). PI 3-kinase, which activates
PKB/Akt, GSK-3, a major target of Akt, and the catalytic domain of PP2A,
a phosphatase that inactivates the PI 3-kinase/PKB pathway, remain unchanged
in their protein expression levels compared with WT controls.
In addition, the observed increases in GLUT-4 and PKB/Akt expression levels are limited to the adipocyte, because skeletal muscle samples do not show any changes in either GLUT-4 or PKB/Akt protein levels (Fig. 8, A and B).
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Insulin-stimulated phosphorylation is diminished selectively in Cav-1
null adipose tissue. As shown in Fig.
3A, using Cav-1 null mice, we demonstrated a 90%
selective reduction in insulin receptor protein levels and a corresponding
decrease in insulin-stimulated phosphorylation of this receptor in the
adipocyte, whereas insulin receptor protein levels remained unchanged in the
liver and muscle. To demonstrate that the liver and muscle tissue of Cav-1
null mice are capable of transmitting insulin signals normally, we again
stimulated mice with insulin (7.5 min, 1 U/Kg) and probed these tissues for
phosphorylated IR-
. As shown in Fig.
9A, liver and muscle tissues showed equivalent IR-
phosphorylation in both WT and Cav-1 null mice, whereas IR-
phosphorylation, along with IR-
itself, were again diminished in the
perigonadal fat pad. These results provide strong evidence that reduced
insulin signaling occurs selectively in Cav-1 null adipose tissue.
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To determine whether the reduced insulin receptor protein and
phosphorylation levels have any functional consequence on insulin signaling in
the adipocyte, we next examined the activation state of two downstream
signaling molecules, PKB/Akt and GSK-3. PKB/Akt is normally activated by
phosphorylation on serine 473 in response to insulin stimulation. In contrast,
GSK-3
is controlled by dual phosphorylation. Phosphorylation of tyrosine
216 leads to activation, whereas inactivation occurs 1) by
phosphorylation of serine 9, by PKB/Akt, and 2) by dephosphorylation
of tyrosine 216 (7). Thus
GSK-3
is normally dephosphorylated on tyrosine 216 in response to
insulin stimulation.
Interestingly, we found that, after insulin treatment, phospho-PKB/Akt
levels were greatly reduced in Cav-1 null adipose tissue compared with WT
controls (Fig. 9B).
Furthermore, in Cav-1 null adipose tissue, phospho-GSK-3 (Y216) levels
failed to decrease after insulin administration
(Fig. 9B). These
results indicate that the reduced insulin receptor protein levels and
decreased activation of the insulin receptor lead to an overall diminishment
of downstream insulin signaling events, as predicted.
Recombinant expression of caveolin-1 in Cav-1 null fibroblasts rescues insulin receptor expression. Our in vivo results clearly show that in the absence of caveolin-1, the insulin receptor protein was found in diminished quantity selectively in the adipocyte (see Fig. 3). These results suggest that the insulin receptor is somehow normally stabilized in the presence of caveolin-1.
To further explore the supporting role that caveolin-1 appears to play for the insulin receptor, we next moved to cell culture systems to examine whether recombinant expression of caveolin-1 can rescue or upregulate insulin receptor expression in caveolin-deficient cells. For this purpose, we chose to use two different fibroblastic cell lines, HEK-293 cells, which express extremely low levels of endogenous caveolin-1, and 3T3 fibroblasts derived from WT and Cav-1 null mouse embryos (28, 42).
To determine whether MEFs provide a good model for dissecting the
mechanisms underlying our findings in whole animals, we first compared WT and
Cav-1 null MEFs for insulin receptor content by Western blot analysis. As
shown in Fig. 10A,
Cav-1 null MEFs contained significantly less IR- protein compared with
WT MEFs, in accordance with our findings using adipose tissue.
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We next transiently expressed caveolin-1 in both MEFs and HEK-293 cells.
Thirty-six hours posttransfection, the cells were lysed and analyzed via
Western blot for caveolin-1 and insulin receptor expression. As shown in
Fig. 10, B and
C, we found that transient expression of caveolin-1
dramatically increased the amount of IR- present in both Cav-1 null 3T3
MEFs and HEK-293T cells.
Virtually identical results were also obtained by immunofluoresence
analysis. Cav-1 null 3T3 fibroblasts transfected with the caveolin-1 cDNA
showed a marked increase in insulin receptor immunostaining, whereas
neighboring untransfected cells revealed very low or undetectable levels of
IR- immunostaining (Fig.
10D).
Thus our findings indicate that caveolin-1 has a dramatic effect on insulin receptor protein levels in both fibroblasts and adipocytes. These data are consistent with the well-accepted notion that fibroblasts are adipocyte precursors during the process of adipocyte differentiation.
The insulin receptor is degraded by the proteasomal pathway. To gain insight into the mechanism by which loss of caveolin-1 could lead to a reduction in insulin receptor protein levels, we next focused on the cellular degradative machinery. Specifically, we chose to examine the role of the proteasomal pathway using the well-known inhibitor MG-132 (28). Cav-1 null MEFs were treated with 1 µM MG-132 over a time course of 6 and 12 h and were then assayed for insulin receptor protein levels via Western blot analysis.
Figure 11 demonstrates a
dramatic rescue of the IR- subunit as early as 6 h posttreatment. This
finding provides further evidence that caveolin-1 indeed stabilizes the
insulin receptor against cellular degradation processes. Caveolin-2 is shown
as a positive control because it has previously been shown to be rescued by
proteasomal inhibition (28),
whereas
-tubulin is shown as a control for equal protein loading.
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Recombinant expression of Cav-1 (61100) in Cav-1
null fibroblasts fails to rescue insulin receptor protein expression. A
multitude of studies have given rise to the idea that caveolin-1 interacts
with numerous proteins via a cytoplasmic NH2-terminal domain
contained within residues 61101, termed the oligomerization domain, or
a region therein (residues 82101), termed the scaffolding domain
(reviewed in Ref. 22). To
further explore the relationship between caveolin-1 and the insulin receptor,
we used an oligomerization/scaffolding domain-deficient caveolin-1 construct,
Cav-1 (
61100), to determine whether this portion of the protein
is necessary for insulin receptor stabilization.
Caveolin-1-deficient MEFs were transiently transfected with either
full-length WT Cav-1 (as in Fig.
11) or Cav-1 (61100). The cells were then lysed and
subjected to Western analysis for IR-
levels. As predicted, the Cav-1
(
61100) mutant failed to rescue insulin receptor levels, unlike
full-length Cav-1 (Fig.
12A). We next used immunofluorescence microscopy to
further evaluate our results from immunoblot analysis. Note that the cell
transfected with Cav-1 (
100)
(Fig. 12B, arrow)
shows no change in insulin receptor immunostaining compared with the
surrounding untransfected cells (arrowheads). See
Fig. 10D for
comparison with WT Cav-1. Thus the above results are consistent with the
notion that the Cav-1 scaffolding domain may play a protective role in
stabilizing the insulin receptor, thereby preventing its proteasomal
degradation.
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DISCUSSION |
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Next, by transient transfection of Cav-1 null 3T3 fibroblasts, we directly
demonstrated that replacement of caveolin-1 is sufficient to rescue insulin
receptor protein expression. Using the well-established inhibitor MG-132, we
showed that in the absence of caveolin-1, the insulin receptor undergoes
proteasomal degradation. Finally, we provided evidence that the
caveolin-scaffolding domain is required to stabilize the insulin receptor,
because the Cav-1 (61100) mutant failed to rescue insulin
receptor expression in Cav-1 null MEFs. Using a cell-permeable peptide
corresponding to the caveolin-1 scaffolding domain
(15), we were able to
demonstrate rescue of the insulin receptor in Cav-1 null MEFs, indicating that
the scaffolding domain by itself is sufficient to functionally stabilize the
insulin receptor (data not shown). Our results show that caveolin-1 is indeed
an important positive regulator of insulin signaling, because it normally
serves to stabilize the insulin receptor at the protein level in both
fibroblasts and adipocytes. These studies are consistent with previous in
vitro studies that demonstrated a direct interaction between caveolin-1 and
the insulin receptor kinase domain
(49).
Pathways of metabolic control are delicately balanced, and it is now clear that caveolin-1 plays an integral part in maintaining normal lipid homeostasis. Lipid balance is regulated by hormonal control in response to an ever changing environment. After a meal, insulin is released, having a multitude of effects on various tissues. In the adipocyte, regarding lipid compounds, insulin serves to activate the production of lipids in their storage form while inhibiting their breakdown. In Cav-1 null mice, it seems that this regulatory step is imbalanced or skewed toward overactive lipolysis. This is consistent with the idea that caveolin-1 functions to 1) enhance insulin mediated lipogenic signals while 2) suppressing the opposing PKA mediated lipolytic signals in the adipocyte (1, 13, 33, 34). This inhibition is due to both a direct negative effect of caveolin-1 on PKA and an indirect effect on PKA via augmentation of anti-lipolytic insulin signals (30). Insulin inhibits lipolysis by activating phosphodiesterase-3, which leads to a decrease in intracellular cAMP levels and a subsequent loss of activation of PKA (47). Insulin also causes a stable inhibitory complex to form between hormone sensitive lipase and lipotransin, the main lipolytic hormone in the adipocyte (43). In our study, we found that Cav-1-null mice have a blunted response to insulin and, presumably, an inability to inactivate PKA-mediated lipolysis.
The role that we have suggested for caveolin-1 in the regulation of
insulin-dependent signals and the development of an insulin-resistant state is
in line with the previously proposed role of caveolin-1 as an activator of
insulin signaling in HEK-293 cells
(49). In their report,
Yamamoto et al. demonstrated that transient transfection of the cDNAs encoding
full-length caveolin-1 or caveolin-3 proteins into HEK-293 cells was
sufficient to increase IRS-1 phosphorylation following insulin stimulation.
Furthermore, they demonstrated a direct interaction between the insulin
receptor kinase domain and the caveolin-scaffolding domain of both caveolin-1
and caveolin-3. More specifically, the scaffolding domain of caveolin-1
(residues 82101) binds to a specific motif in the kinase domain of the
insulin receptor
(1193WSFGVVLW1200;
X
XXXX
, where
represents an aromatic amino acid)
(4,
49). Thus they proposed that
this caveolin-1/IR-
interaction somehow mediates the activation of the
insulin receptor. Our new in vivo findings (described here) support this idea
and show that the increased IRS-1 phosphorylation that Yamamoto et al.
observed is most likely due to an increase in the insulin receptor protein
itself, via a stabilizing interaction with caveolin-1. In further support of
our findings, it has been shown that treatment with the lipid
raft/caveolae-disrupting agent, methyl-
-cyclodextrin, inhibits insulin
and insulin-like growth factor-1-induced activation of IRS-1 and the
downstream activation of PKB/Akt
(25,
26). Furthermore, these
authors demonstrated that cells treated with methyl-
-cyclodextrin become
insulin-resistant, in that they are no longer capable of glucose uptake. Our
in vivo findings are consistent with these reports showing that insulin
stimulation of Cav-1-deficient adipose tissue results in diminished activation
of the insulin receptor and its downstream targets.
In another study, Nystrom et al. (21) systematically mutated the caveolin-binding motif within the insulin receptor kinase domain (W1193G/F1195G/W1200G). When transfected into COS-7 cells, they found that these caveolin-binding motif mutants were poorly expressed, compared with the WT insulin receptor (21). Together with our findings, this lack of insulin receptor expression demonstrates the critical need for a functional interaction between caveolin-1 and the insulin receptor to achieve normal receptor levels in certain cell types.
This idea can be further applied to the clinical setting. A subset of patients with severe insulin resistance has mutations within the caveolin-binding motif of the insulin receptor (W1193L and W1200S) (9, 10, 12, 17, 18, 35). These caveolin-binding motif mutations lead to enhanced degradation of the insulin receptor (12, 35) (Table 1). Thus a better understanding of the interaction between caveolin-1 and the insulin receptor may lead to the development of new therapies that functionally stabilize insulin receptor protein expression.
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In this and our previous study demonstrating adipose tissue abnormalities
(27), we found that Cav-1 null
mice maintain relative euglycemia in the face of insulin resistance. To
explain these findings, we examined the potential compensatory role of the
IGF-1R. We found that there is no change in the amount of IGF-1R protein
levels in Cav-1 null adipose tissue, eliminating the IGF-1R as a major
compensatory player. Therefore, a major impairment of insulin signaling at the
level of the fat cell in Cav-1 null mice does not lead to the classic early
manifestations of impaired glucose tolerance, i.e., normoglycemia associated
with fasting hyperinsulinemia. Interestingly, in contrast to the generally
decreased levels of GLUT-4 in obese and insulin-resistant models
(40), we found an upregulation
of the insulin-responsive glucose transporter, GLUT-4, that at least in part
may explain the ability of Cav-1 null mice to sustain euglycemia despite local
insulin resistance. Most importantly though, insulin receptor levels remain
normal in the skeletal muscle tissue of Cav-1 null mice. Because 75% of
glucose is normally disposed of in skeletal muscle tissue, it is possible that
the skeletal muscle compensates for adipose tissue in this regard
(33). Also, in support of our
findings, it is interesting to note that both the recently developed
adipose-tissue selective insulin receptor knockout mouse (FIRKO) and the
combined muscle and adipose tissue insulin receptor knockout mouse maintain
euglycemia (2,
14).
Many recent studies have demonstrated that perhaps the most important determinant of insulin sensitivity is the lipid content of muscle and liver tissue. In the case of obesity or lipoatrophy, the adipocyte is unable to store lipids, and thus, triglycerides accumulate in muscle and liver. This leads to severe insulin resistance in these tissues and the eventual development of type II diabetes. Disease progression can be halted or even reversed in some cases when triglycerides, particularly in the muscle, are eliminated by oxidation. Recent evidence points to two adipokines, leptin and Acrp30, as being instrumental in this process. Leptin directly activates AMP-activated protein kinase in the muscle, which leads to fatty acid consumption, via oxidation, and the subsequent improvement of insulin sensitivity (8, 16, 41, 50). Similarly, Acrp30 has been reported to be involved in activating expression of molecules responsible for fatty acid transport and combustion in skeletal muscle (50). Thus it seems that the adipocyte is the primary player in the progression of insulin resistance, with the muscle and liver being the determinants of how far the disease will progress. Given that liver and skeletal muscle express normal levels of insulin receptor in Cav-1 null mice, this could explain why we observed euglycemia and a relatively mild postprandial hyperinsulinemia in Cav-1 null animals.
Recently, using the Cre-loxP system, Blüher and colleagues (2) developed a FIRKO mouse. Because this mouse lacks the insulin receptor specifically in adipocytes, as does the Cav-1 null mouse, a discussion of this model is highly relevant. Important similarities between these two mouse models include resistance to age-related obesity, diminished body fat, and normal fasted and fed glucose levels on a chow diet, as well as normal fed insulin levels on a chow diet. However, these two mice differ substantially on many other parameters. Most related to the work reported here, FIRKO mice respond normally to an ITT even at 10 mo, an age when WT mice show significant insulin resistance. The authors claim (2) that this represents protection against obesity-related insulin intolerance, because their mice are lean at this age. However, Cav-1 null mice display a markedly blunted response to insulin at 5 mo of age, even though Cav-1 null mice are also resistant to age-related obesity. Other pertinent differences between these two mouse models include decreased brown fat mass in FIRKO mice (which is dramatically increased in Cav-1 null mice), increased plasma leptin and Acrp30 levels in FIRKO mice (which are decreased in Cav-1 null mice), and reduced serum triglyceride levels in FIRKO mice (which are markedly elevated in Cav-1 null mice). Several possibilities exist to explain these differences. Most importantly, the loss of caveolin-1 may have pleiotropic effects on multiple organ systems, signaling cascades, and cellular functions, the extent of which are just now being explored. Hosts of signaling molecules are known to localize to and signal through caveolae, only one of which is the insulin receptor. Therefore, it would be expected that dysregulation of many of these different cascades would lead to multiple independent, yet overlapping phenotypes. Yet, our results clearly demonstrate that caveolin-1 plays a significant role in insulin signal transduction via stabilization of the receptor.
The degree of insulin resistance found in Cav-1 null mice does not result in fasting hyperinsulinemia. This is in contrast to conventional models of type II diabetes. Similar findings have recently been observed in the FIRKO mouse. The FIRKO mice show no fasting hyperinsulinemia despite significant tissue-specific insulin resistance, as evidenced by impaired insulin-dependent glucose uptake in isolated adipocytes (2). However, the FIRKO mouse does not display whole body fasting or postprandial hyperinsulinemia. In fact, it maintains normal or below average insulin levels throughout its life. Thus the FIRKO mouse model indicates that insulin resistance in adipose tissue per se does not lead to fasting hyperinsulinemia. In light of this, the finding that Cav-1 null mice only display postprandial hyperinsulinemia is very much in line with the observations made in the FIRKO mouse model.
It is important to note that Cav-1 null mice are functionally deficient in
caveolin-2 as well. Caveolin-1 and -2 form a functional heterooligomeric
complex in cells where they are coexpressed
(36). Caveolin-2 requires
coexpression with caveolin-1; in the absence of caveolin-1, caveolin-2 remains
trapped in the Golgi complex and undergoes proteasomal degradation
(24). Therefore, all of the
phenotypes identified in the Cav-1-null mouse must also be examined in the
Cav-2-null mouse to determine whether the findings are due to a functional
caveolin-2 deficiency. Importantly, we have recently shown that Cav-2-null
mice do not show any lipid imbalances or adipose tissue atrophy, indicating
that the adipose tissue abnormalities observed in Cav-1-null mice are indeed
due to the loss of caveolin-1 expression and are not caveolin-2 dependent.
Consistent with these findings, we showed here that Cav-2-null mice express
normal levels of IR- in their adipose tissue. Thus caveolin-2 expression
is not required for maintaining normal insulin receptor expression levels in
the adipocyte.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health (NIH), Muscular Dystrophy Association, American Heart Association, and Breast Cancer Alliance (all to M. P. Lisanti) and by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-55758 and a grant from the American Diabetes Association (to P. E. Scherer). A. W. Cohen, B. Razani, and T. M. Williams were supported by NIH Medical Scientist Training Grant T32-GM-07288. M. P. Lisanti is the recipient of a Hirschl/Weil-Caulier Career Scientist Award. T. P. Combs was supported by NIH Hormones/Membrane Interactions Training Grant T32-DK-07513-15.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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