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J. Biol. Chem., Vol. 278, Issue 35, 33377-33383, August 29, 2003
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From the Department of Cellular and Molecular Physiology, Joslin Diabetes Center, Harvard Medical School, One Joslin Place, Boston, Massachusetts 02215
Received for publication, March 25, 2003 , and in revised form, June 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
, and 
and the peroxisome
proliferator-activated receptor gamma (PPAR
). Expression and activation
of these transcription factors are required for the expression of adipogenic
markers such as fatty acid synthase (FAS) and the insulin-sensitive glucose
transporter Glut4.
Upstream signals regulating the expression and activation of these transcription factors during adipocyte differentiation are not fully understood. However, the derivation of white and brown adipocyte cell lines that are deficient in insulin or insulin-like growth factor I (IGF-I) signaling genes have provided useful tools for determining the contribution of the insulin/IGF-I signaling pathways to both adipogenesis and differentiation-dependent signaling. Pre-adipocyte cell lines derived from IRS-1-deficient cells are impaired in their ability to differentiate into mature adipocytes and show defects in lipid synthesis (1, 2). Although IRS-2-deficient adipocytes show only modest defects in adipogenesis, insulin-stimulated Glut4 translocation and glucose uptake in mature adipocytes is dramatically reduced (3). In addition, pharmaceutical inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of dominant negative mutant subunits abrogates differentiation of both white and brown adipocytes in vitro (1, 4, 5). Although these signaling components are downstream of both insulin and IGF-I, it is currently hypothesized that IGF-I is the more essential inducer of adipocyte differentiation, because the IGF-I receptor (IGF-IR) is the predominant receptor in preadipocytes, and high levels of insulin are required for differentiation, presumably by activity through IGF-IR. However, mice with either a total fat-specific (FIRKO) or a brown fat-specific (BATIRKO) inactivation of the insulin receptor have reduced fat mass compared with their wild type littermates (6, 7). These data suggest that insulin action through the insulin receptor is a critical factor in the regulation of adipogenesis.
Insulin receptor (IR) mRNA is alternatively spliced, creating two isoforms
that either contain (IRB) or lack (IRA) exon 11, which
encodes 12 amino acids at the carboxyl terminus of the IR -subunit.
Functional studies have consistently shown that IRA has a 2-fold
higher affinity for insulin. Additionally, IGF-II preferentially binds
IRA, and IRA may have a higher internalization rate than
IRB
(8–11).
Expression of the IR splice isoforms is regulated both developmentally and in
a tissue-specific manner in mammals
(12–14).
Despite the variable expression of IR isoforms in insulin-sensitive tissues,
such as liver, muscle, and white fat, the regulation of splicing and function
of these isoforms in brown fat is not understood.
In the current study we have investigated the direct role of the insulin receptor and its two isoforms in brown fat adipogenesis by generating immortalized brown pre-adipocytes from mice lacking the insulin receptor (FIRKO) and their control (IRlox) littermates, as well as IR-deficient and normal preadipocytes that overexpress IR. We find that brown adipocytes undergo IR isoform switching during differentiation and that there is a critical need for the proper level of insulin receptors to initiate adipocyte differentiation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, anti-C/EBP, and anti-UCP1 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA); anti-Glut4 (Chemicon International,
Inc., Temecula, CA); and anti-phospho-AKT and anti-phospho-MAPK (New England
BioLabs, Beverly, MA). Protein A-Sepharose was purchased from Amersham
Biosciences (Piscataway, NJ), and [-32P]ATP was from
PerkinElmer Life Sciences (Boston, MA). Phosphoinositol was obtained from
Avanti Polar-Lipids, Inc. (Alabaster, AL), polyvinylidene difluoride membrane
was from Fisher Scientific (Pittsburgh, PA), thin-layer chromatography plates
were from VWR (Bridgeport, NJ), and electrophoresis supplies were from Bio-Rad
Laboratories (Hercules, CA). Troglitazone was a gift from Warner-Lambert Co.
(Ann Arbor, MI). All other supplies, unless indicated, were purchased from
Sigma Chemical Co. (St. Louis, MO).
Cell Isolation and Culture—Cells that were homozygous for a
floxed allele of exon 4 of the insulin receptor (IRlox) were used
as controls for all studies. Brown pre-adipocytes were isolated from newborn
control IRlox and FIRKO mice by collagenase digestion as described
previously (15).
Pre-adipocytes were immortalized by infection with a pBABE retrovirus encoding
SV40 T-antigen and selected with 2 µg/ml puromycin. Cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% v/v fetal calf
serum in a 5% CO2 environment. For in vitro recombination
of the insulin receptor, pre-adipocytes harboring a floxed allele of the
insulin receptor (IRlox) were first plated at a subconfluent
density. After 24 h, cells were infected with an adenovirus encoding
cre recombinase at a titer of 109 plaque-forming units.
After 1 h the viral supernatant was replaced with culture medium. Individual
colonies were selected for IR recombination using PCR with genomic DNA
(16). To differentiate cells,
pre-adipocytes were first grown to confluence in culture medium containing 20
nM insulin and 1 nM triiodothyronine (differentiation
medium). Adipogenesis was induced by treating confluent cultures (day 0) with
differentiation medium supplemented with 0.5 mM
isobutylmethylxanthine, 0.5 µM dexamethasone, and 0.125
mM indomethacin (induction medium) for 48 h. After this induction
phase (day 2), cells were returned to differentiation medium, which was
replenished every 2 days until day 6 when cells were considered mature
adipocytes. For activation of PPAR, 25 µM troglitazone
was included in the induction medium for 48 h.
Oil Red O Staining—Dishes were washed once with phosphate-buffered saline and fixed with 10% buffered formalin for at least 16 h at 4 °C. Cells were then stained for 4 h at room temperature with oil red O solution (5 g/liter in isopropyl alcohol), washed three times with water, and visualized.
Plasmids and Retroviral Infection—The PPAR retroviral
expression vector has been described previously
(1). Coding sequences for the
individual splice isoforms of the human IR either containing or lacking exon
11 (kindly provided by Ingo Leibiger, Karolinska University, Stockholm,
Sweden) were cloned into pBABE-bleo or -hygro vectors. Subconfluent 
NX
packaging cells were transfected by LipofectamineTM reagent (Invitrogen,
Carlsbad, CA) with 10 µg of retroviral vector, and viral supernatants were
collected 48 h after transfection. Control or IRKO pre-adipocytes were
infected with Polybrene (4 µg/ml)-supplemented virus for 48 h and then
placed in selection medium containing either the bleomycin analog zeocin (250
µg/ml) or hygromycin (200 µg/ml, Invitrogen).
RNA Extraction and Semi-quantitative RT-PCR—Total RNA was
isolated using the single-step method of Chomczynski and Sacchi
(17). To analyze gene
expression by PCR, 1 µg of total RNA was primed with oligo(deoxythymidine)
in the presence of murine mammary tumor virus reverse transcriptase
(Invitrogen) to synthesize cDNA. The samples were diluted 5-fold, and 5% of
the total volume was used for subsequent PCR. Primers and PCR conditions are
as follows: mouse IR exon 4 primer 1,
5'-ACTGTTCGGAACCTGATGACCC-3'; primer 2,
5'-GTGATACCAGAGCATAGGAGCG-3'; mouse IR exon 11 primer 1,
5'-ATCAGAGTGAGTATGACGACTCGG-3'; primer 2,
5'-TCCTGACTTGTGGGCACAATGGTA-3'; human IR exon 11 primer 1,
5'-ACCAGAGTGAGTATGAGGATTCGG-3'; primer 2,
5'-TCCGGACTCGTGGGCACGCTGGTC; Glut4 primer 1,
5'-GAGATACTCATTCTTGGACGG-3'; primer 2,
5'-ACAGCATTGATGCCTGAGAGC-3'; PPAR primer 1,
5'-GAATACCAAAGTGCGATCAAAGTA-3'; primer 2,
5'-CCAAACCTGATGGCATTGTGAGAC-3'; glyceraldehyde-3-phosphate
dehydrogenase primer 1, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3'; primer
2, 5'-CATGTAGGCCATGAGGTCCACCAC-3'. IR exon 11 and PPAR PCRs
were performed using PCR buffer (Invitrogen) supplemented with 1.5
mM MgCl2 at 93 °C for 30 s, 60 °C for 30 s, and
72 °C for 45 s for 27 cycles. IR exon 4, Glut4, and
glyceraldehyde-3-phosphate dehydrogenase PCRs were performed at 93 °C for
45 s, 56 °C for 45 s, and 72 °C for 1 min for 40 cycles (IR exon 4) or
27 cycles (Glut4 and glyceraldehyde-3-phosphate dehydrogenase). Cycle
conditions were optimized within the linear range of detection to reflect
relative levels of expression. Reaction products were resolved on 2% agarose
gels.
Cell Lysis, Immunoblotting, and Immunoprecipitation—Cells were lysed in extraction buffer containing 50 mM HEPES, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM NaP2O7, 10 mM NaF, 2 mM EDTA, 10% glycerol, 1% Igepal CA-630, 1 mM sodium orthovanadate, 10 µg/ml trypsin inhibitor, 0.5 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride. Extracts were sonicated and clarified by centrifugation for 20 min, and protein concentrations were determined by the Bradford method (18). For immunoblotting of whole cell lysates, equal amounts of protein were directly solubilized in Laemmli sample buffer and resolved on SDS-containing polyacrylamide gels. Protein was transferred to polyvinylidene difluoride membranes and immunoblotted using the indicated primary antibodies. Blots were incubated with the appropriate peroxidase-conjugated secondary antibody and developed with enhanced chemiluminescence. For immunoprecipitations, 500 µg of protein was incubated with the indicated antibodies and protein A-Sepharose for 16 h at 4 °C. Immune complexes were collected by centrifugation, washed in extraction buffer, solubilized in Laemmli sample buffer, and analyzed as above.
PI3K Assays—Assays were completed as previously described (1) with the following modifications. Reactions were terminated by the addition of 150 µl of chloroform:methanol:12 N HCl (100:200:2, v/v). The organic phase was washed once with methanol:1 N HCl (1:1) and concentrated by centrifugation under vacuum. The lipids were resolved with chloroform and spotted onto a silica gel thin-layer chromatography plate. The plate was developed in CHCl3:MeOH:H2O:NH4OH (120:94: 23:2.4, v/v), dried, exposed to a PhosphorImager screen, and quantitated with a Molecular Dynamics densitometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To ascertain the role of the insulin receptor in differentiation, control
IRlox and IRKO pre-adipocytes were differentiated into brown
adipocytes using insulin, triiodothyronine, isobutylmethylxanthine,
dexamethasone, and indomethacin as described under "Experimental
Procedures." At the end of the differentiation protocol (day 6), cells
from both genotypes were stained using the fat-specific dye oil red O
(Fig. 2A). In control
IRlox cells, 90–100% of cells accumulated multiocular fat
droplets. This was coordinated with an induction of various adipogenic mRNAs
such as PPAR and Glut4. In control adipocytes, splicing of exon 11 of
the insulin receptor mRNA was also regulated in a differentiation-dependent
manner (Fig. 2B). In
pre-adipocytes (day 0) the level of insulin receptor mRNA was low and
predominantly the A isoform. During differentiation there was an increase in
the total levels of IR mRNA with a coordinated increase in the B isoform of IR
such that the ratio of IRB:IRA isoform was 
60%:40%
by day 6. As has been shown previously
(1), during differentiation
there was an increase in mRNA levels of the adipogenic markers PPAR and
Glut4 in control cells.
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Insulin Receptor-deficient Pre-adipocytes Fail to
Differentiate—In comparison to controls, no fat droplets were
observed in either type of IRKO pre-adipocytes after the differentiation
protocol (Fig. 2A).
This failure to accumulate lipid was observed in ten independent IR-deficient
cell lines (data not shown) and was accompanied by an inability to induce the
differentiation markers PPAR and Glut4
(Fig. 2B). Although IR
mRNA was still present in IRKO cells, because deletion of exon 4 by
cre recombination creates a frameshift mutation predicting a
premature stop codon, no mature IR protein was present in these cells (see
above). In parallel with the lack of differentiation, IRKO cells also showed
no change in the splicing of IR mRNA during time in culture. Similarly, the
increased expression of PPAR and Glut4 was not observed in IRKO
cells.
Reconstitution of IR in IRKO Cells Fails to Restore
Differentiation—To determine whether either of the IR splice
isoforms has a unique role in adipogenesis, IRKO cells were infected with a
retrovirus encoding for either human IRA or IRB.
Surprisingly, IRKO cells reconstituted with either IR isoform failed to
differentiate as measured by oil red O staining
(Fig. 2C) or
accumulation of the adipogenic markers PPAR and Glut4
(Fig. 2D). These
results were verified in three independent IRKO cell lines. Coexpression of
both splice isoforms also failed to restore differentiation in IRKO cells
(data not shown).
To ensure that the re-expressed IR isoforms were functional proteins, control, IRKO, and IR-reconstituted KO pre-adipocytes were stimulated with insulin, and phosphorylation of downstream targets was analyzed (Fig. 3). IR protein expression was very low in control IRlox pre-adipocytes, but was undetectable in IRKO cells. IR-reconstituted KO cells showed dramatically increased IR protein levels and a markedly increased level of IR tyrosine phosphorylation following insulin stimulation. Insulin was also able to stimulate phosphorylation of AKT in control and IR-reconstituted KO cells, but not in IRKO cells. Therefore, IR is required for insulin-stimulated AKT phosphorylation. Surprisingly, despite the absence of insulin receptors, insulin was able to induce phosphorylation of MAPK in IRKO cells, although phosphorylation was slightly enhanced in cells re-expressing IR. Because IRKO cells lack insulin receptors, this increase in MAPK phosphorylation in IRKO pre-adipocytes most likely occurs via insulin action through the IGF-I receptor. This result was not due to receptor compensation, because IGF-I receptor levels were not different between the cell lines (data not shown). Both splice isoforms of IR (hIRA and hIRB) were equivalent in their ability to activate downstream signaling molecules.
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Phosphorylation of IR, IRS-1, and IRS-2 during Adipogenesis Is Impaired
in IRKO Cells—A number of proteins implicated in insulin signaling
are modified during differentiation. To determine the contribution of IR to
these events, tyrosine phosphorylations of IR, IRS-1, and IRS-2 were examined
in control IRlox-, IRKO-, and IR-reconstituted KO cells during the
differentiation protocol. Because hIRA and hIRB signaled
similarly in these cells (see Fig.
3), only results for cells expressing hIRA are shown
for simplicity. Similar to mRNA levels, protein expression of IR was low in
control pre-adipocytes but was increased in mature adipocytes
(Fig. 4). IRKO cells failed to
express mature IR protein. IR-reconstituted KO cells showed an abundant
increase in expression of IR in pre-adipocytes (day 0). Although these levels
were maintained throughout differentiation, the absolute level of IR was only
3-fold higher at day 6 compared with control adipocytes.
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Following induction of differentiation, tyrosine phosphorylation of IR was elevated at day 2 in control cells. At days 4–6 of differentiation, levels of IR tyrosine phosphorylation declined even though total IR protein levels were elevated. As expected, IRKO cells showed no increase in IR tyrosine phosphorylation. In IR-reconstituted KO cells tyrosine phosphorylation of IR was faintly detected at day 0 but was dramatically increased at day 2 after induction of differentiation. Following the pattern of IR tyrosine phosphorylation, both IRS-1 and IRS-2 were tyrosine-phosphorylated at day 2 of differentiation in control cells. This phosphorylation was absent in IRKO cells and dramatically reduced in IR-reconstituted KO cells despite the high levels of IR tyrosine kinase activity.
Reconstitution of IR Expression in IRKO Cells Restores PI3K Activation
and MAPK Dephosphorylation—Inhibition of PI3K activity by
pharmaceutical inhibitors or expression of dominant-negative subunits disrupts
adipocyte differentiation (1,
4), indicating PI3K activity is
essential for the adipogenic program. To determine whether PI3K activation was
directly coupled to differentiation, PI3K activity in phosphotyrosine
immunoprecipitates was analyzed in the three cell lines. Similar to the
pattern of IRS-1 and IRS-2 tyrosine phosphorylation, PI3K activity was induced
4-fold at day 2 and then returned to basal levels at days 4 and 6 in
control cells (Fig.
5A). There was no induction of PI3K activity in IRKO
cells. In IR-reconstituted KO cells basal PI3K activity was higher, and there
was a further induction at day 2 of differentiation. Thus, although the
insulin receptor appears to be required for the PI3K activation observed
during differentiation, activation of PI3K, in the presence of an
inappropriate level of IR, is not sufficient to drive the adipogenic
program.
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To investigate signaling further downstream of IR, IRS, and PI3K, phosphorylation of AKT and MAPK was examined. In control cells, there was a transient increase in AKT phosphorylation at day 2 of differentiation that paralleled PI3K activation (Fig. 5B, upper panel). Phosphorylation of AKT was absent in IRKO cells. IR-reconstituted KO cells showed an increase in AKT phosphorylation at day 0, which declined during differentiation. MAPK activity has been associated with inhibition of differentiation (21, 22), and in accordance with this, MAPK phosphorylation was elevated in control pre-adipocytes (day 0) but then decreased during differentiation (Fig. 5B, lower panel). By contrast, MAPK phosphorylation in IRKO cells was high and remained elevated throughout the culture period, correlating with the inability of these cells to differentiate. Phosphorylation of MAPK in IR-reconstituted KO cells resembled the pattern observed in control cells, i.e. phosphorylation was elevated in pre-adipocytes and declined at the later days of the differentiation program. However, as noted above, these cells failed to differentiate.
PPAR Expression and Activation Partially Restores
Differentiation in IRKO Cells—The finding that IR-reconstituted KO
cells were similar to control cells in their ability to alter
differentiation-dependent signaling such as activation of PI3K and
down-regulation of MAPK phosphorylation, but failed to differentiate as
measured by oil red O staining or accumulation of adipogenic markers such as
PPAR and Glut4, suggested that the block in adipogenesis in these cells
may occur at a later stage in differentiation.
To test this hypothesis, the adipogenic transcription factor PPAR
was introduced into IRKO cells via retroviral-mediated gene transfer. To
enhance PPAR activation, some experiments were also performed with the
addition of the PPAR agonist troglitazone. Differentiation was measured
by the induction of the adipogenic proteins PPAR, Glut4, FAS, and
C/EBP (p42 and p30 products). As described above, fully differentiated
control cells showed high expression of all the measured adipogenic markers
(Fig. 6, lane 1).
Addition of PPAR or troglitazone in control cells exhibited no
significant changes in protein expression, presumably due to the natural
differentiation potential of these cells
(Fig. 6, lanes
2–4). In contrast with the results in IRS-1 knockout cells
(1), expression of PPAR
or its activation by troglitazone only slightly improved the ability of IRKO
cells to differentiate as measured by a modest increase in the levels of
endogenous PPAR, Glut4, and FAS
(Fig. 6, compare lanes
6 and 7 to lane 5). The combination of PPAR
expression with activation by troglitazone further enhanced differentiation in
IRKO cells (Fig. 6, compare
lane 8 with lanes 6 and 7). However, under these
conditions differentiation of IRKO cells was still markedly reduced compared
with control cells. To determine whether PPAR expression and IR
signaling could coordinately enhance differentiation, human insulin receptor
(hIRA, lanes 9 and 10; or hIRB,
lanes 11 and 12) was reintroduced into IRKO cells expressing
PPAR. Expression of either IR splice isoform was able to prevent
adipogenesis induced by moderate overexpression of PPAR in IRKO cells
in the presence or absence of troglitazone
(Fig. 6, compare lanes
10 and 12 to lane 8). These data support the hypothesis
that high levels of IR may be inhibitory to adipogenesis.
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Overexpression of IR in Control Pre-adipocytes Inhibits
Differentiation—To determine whether high IR expression disrupts
adipogenesis, the human insulin receptor or an empty vector (+pBABE) was
introduced into control IRlox cells
(Fig. 7). Similar to uninfected
cells, vector-infected cells accumulated multiocular fat droplets as measured
by oil red O staining (Fig.
7A). However, fat accumulation was not detected in cells
overexpressing either isoform of IR. This defect in differentiation was
mirrored by the failure of these cells to induce expression of the adipogenic
markers PPAR, Glut4, FAS, C/EBP , and UCP-1, whereas control and
vector-infected cells showed a robust induction of these proteins by day 6 of
differentiation (Fig.
7B).
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Because IRKO cells reconstituted with IR failed to differentiate but
exhibited some differentiation-dependent signaling (see above), PI3K
activation was examined in control cells that overexpress IR. As shown above,
PI3K activity associated with phosphotyrosine increased 4-fold at day 2
in control cells (Fig.
5C). Although basal PI3K activity was higher in cells
overexpressing IR, a similar maximal level of activity was observed at day 2
of differentiation. The pattern of PI3K activation was paralleled by
phosphorylation of AKT, a downstream target
(Fig. 5D, upper
panel). In cells overexpressing IR, AKT phosphorylation was initially
high at the beginning of the differentiation program (days 0 and 2) but
decreased thereafter. MAPK phosphorylation was also elevated in pre-adipocytes
(Fig. 5D, lower
panel). This phosphorylation was absent during the middle phases of the
differentiation protocol but was increased again by day 6. Control cells that
overexpress IR fail to differentiate but maintain insulin signaling through
PI3K, AKT, and MAPK.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Expression of either IRA or IRB restores insulin
signaling in IR-deficient pre-adipocytes. In contrast to observations in
pancreatic cells (25),
both receptors are equally capable of initiating phosphorylation of AKT, MAPK,
or p70S6K (data not shown) in response to insulin. However, we were unable to
examine the individual contribution of each isoform in mature adipocytes where
expression of both receptors occurs naturally.
Using standard differentiation protocols, we observed that insulin
receptor-deficient brown pre-adipocytes failed to differentiate as measured by
the lack of lipid accumulation or the expression of the adipogenic markers
PPAR, FAS, and Glut4. The requirement for the insulin receptor in
adipocyte differentiation in vitro is supported by gene dosage
studies performed in 3T3-L1 cells. 3T3-L1 fibroblasts with a somatic
inactivation of one allele of the insulin receptor showed a 50–70%
reduction in IR levels and exhibited impaired differentiation with only
30–50% of cells displaying morphological evidence of adipogenesis
(26). Although insulin
receptor levels are low in pre-adipocytes, this minimal expression is required
to initiate the adipogenic program. The failure of IRKO cells to differentiate
also resembles defects seen in IRS-1-deficient cells, reiterating the
importance of insulin signaling in brown fat adipogenesis.
Even though control or IRKO cells overexpressing IR fail to terminally differentiate, some insulin receptor- and differentiation-dependent signaling is preserved in these cells. For example, PI3K activity peaks at day 2 in control cells. Although PI3K activity is absent in IRKO cells, IR reconstitution restores a similar pattern of PI3K activity after the hormonal induction of adipogenesis, suggesting that the insulin receptor is required for this event. PI3K activity is also maintained in control pre-adipocytes that overexpress IR. Previous data has suggested that PI3K activity during the induction phase is essential for adipogenesis in both white and brown fat cell models. Our data would suggest that, although required, PI3K activity is not sufficient to drive adipocyte differentiation.
Previous studies have shown that high MAPK phosphorylation correlates with
the inability to differentiate, and overexpression of MAPK or its upstream
activator MEK1 inhibits 3T3-L1 differentiation, possibly by reducing
PPAR activity (22,
27). In control
pre-adipocytes, MAPK phosphorylation is high then declines with
differentiation. IRKO cells, which fail to differentiate, also fail to
attenuate MAPK phosphorylation. However, IR-reconstituted cells and control
cells overexpressing IR exhibit a decrease in MAPK phosphorylation during the
differentiation program similar to control cells but show similar defects in
adipogenesis. These data suggest that the classic insulin-dependent signaling
induced during the early phases of differentiation can be uncoupled from the
production of the mature adipocyte phenotype.
Unlike IRS-1 reconstitution in IRS-1 KO cells, reconstitution of the insulin receptor in IRKO cells fails to restore differentiation (1). IR-reconstituted KO pre-adipocytes do not accumulate lipid or induce adipogenic proteins when induced to differentiate. This appears to be due to the high level of insulin receptor in the pre-adipocyte stage. This hypothesis is validated by the fact that control pre-adipocytes overexpressing IR also fail to differentiate. Thus it appears that the elevated levels of IR re-introduced in IRKO or control cells upset the balance between IR and IGF-IR that may be required for normal differentiation.
Insulin and IGF-I receptors are highly homologous receptors, each composed
of two -heterodimers linked by disulfide bonds
(28,
29). Individual
-heterodimer subunits from IR and IGF-IR can also combine to form
hybrid receptors (30).
Previous studies have shown that both IGF-I and insulin are able to induce
adipogenic and thermogenic genes in brown adipocytes
(31). Fetal brown adipocytes
possess a number of high affinity IGF-I binding sites and express both IGF-I
and IGF-IR mRNA during development
(32). As noted above, while
insulin receptor expression increases during differentiation, overexpression
of IR inhibits differentiation. In brown preadipocytes and 3T3-L1 fibroblasts,
IR levels are low and IGF-IR is the primary signaling receptor
(33).2
With the induction of differentiation, IR expression increases dramatically
such that it is the predominant receptor in mature adipocytes. Hybrid receptor
formation rises in parallel with the increase in insulin receptor levels. High
IR expression would increase the number of binding sites for insulin through
classic insulin receptors but place more of the IGF-I receptors in hybrid
complexes. We hypothesize that these hybrids are like the insulin receptor in
that they are unable to support differentiation. Thus, the increase in the
insulin signaling pathway indirectly alters the response to the IGF-I
receptor, which adversely affects cell growth or differentiation. Indeed,
Chinese hamster ovary cells overexpressing the insulin receptor are more
sensitive to insulin but become resistant to IGF-I, although the levels of
IGF-IR remain unchanged, suggesting that high levels of IR impair IGF-I
signaling (34). Taken
together, these data suggest that abnormal expression of IR in pre-adipocytes
might disrupt crucial IGF-I signaling required for adipogenesis. Therefore, a
correct balance of IR and IGF-IR must be tightly controlled at all stages of
differentiation. Depletion or inappropriate overexpression of the insulin
receptor early in the process impairs brown adipocyte differentiation. Thus,
the increase in insulin receptor later in adipogenesis may be important for
both insulin regulation of metabolism and the normal differentiation of this
cell type.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 617-732-2635; Fax:
617-732-2487; E-mail:
c.ronald.kahn{at}joslin.harvard.edu.
1 The abbreviations used are: UCP-1, uncoupling protein-1; C/EBP, C/AAT
enhancer binding protein; FIRKO, fat-specific insulin receptor knockout; IRKO,
insulin receptor knockout; PPAR, proliferator-activated receptor gamma;
FAS, fatty acid synthase; IGF-I, insulin-like growth factor I; IGF-IR, IGF-I
receptor; PI3K, phosphatidylinositol 3-kinase; IR, insulin receptor; MAPK,
mitogen-activated protein kinase; MEK1, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase 1.
2 A. J. Entingh and C. Ronald Kahn, unpublished data.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A. Entingh-Pearsall and C. R. Kahn Differential Roles of the Insulin and Insulin-like Growth Factor-I (IGF-I) Receptors in Response to Insulin and IGF-I J. Biol. Chem., September 3, 2004; 279(36): 38016 - 38024. [Abstract] [Full Text] [PDF]
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