From the INSERM U.449 and Human Nutrition Research
Center of Lyon, Faculty of Medicine R. Laennec, Lyon Cédex
08, France, ¶ INSERM Avenir and EA3502, Paris VI University,
Department of Nutrition, Hôtel-Dieu, 75004 Paris,
France, and
Department of Pediatrics and Genetics, Howard
Hughes Medical Institute, Beckman Center, Stanford University School of
Medicine, Stanford, California 94305
Received for publication, January 10, 2003, and in revised form, February 20, 2003
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ABSTRACT |
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Insulin action in target tissues involved precise
regulation of gene expression. To define the set of insulin-regulated
genes in human skeletal muscle, we analyzed the global changes in
mRNA levels during a 3-h hyperinsulinemic euglycemic clamp in
vastus lateralis muscle of six healthy subjects. Using 29,308 cDNA
element microarrays, we found that the mRNA expression of 762 genes, including 353 expressed sequence tags, was significantly
modified during insulin infusion. 478 were up-regulated and 284 down-regulated. Most of the genes with known function are novel targets
of insulin. They are involved in the transcriptional and translational
regulation (29%), intermediary and energy metabolisms (14%),
intracellular signaling (12%), and cytoskeleton and vesicle traffic
(9%). Other categories consisted of genes coding for receptors,
carriers, and transporters (8%), components of the
ubiquitin/proteasome pathways (7%) and elements of the immune response
(5.5%). These results thus define a transcriptional signature of
insulin action in human skeletal muscle. They will help to better
define the mechanisms involved in the reduction of insulin
effectiveness in pathologies such as type 2 diabetes mellitus, a
disease characterized by defective regulation of gene expression in
response to insulin.
Insulin is an anabolic hormone that exerts a wide spectrum of
effects and modulates a variety of biological processes and metabolic
pathways such as glucose and lipid metabolism, protein synthesis and
degradation, cell growth and differentiation, and DNA synthesis. To
perform these actions insulin modifies the activity and the subcellular
location of key regulatory enzymes and proteins, often by affecting
their phosphorylation state (1). Furthermore, insulin also controls the
amount of numerous proteins, in part by acting at the level of mRNA
translation and mainly at the level of their gene expression (2). Over
the past 15 years, the regulation of gene expression by insulin has
been recognized as a major effect of the hormone, and about 150 insulin-regulated genes have been reported (2). However, the
identification of insulin-regulated genes has been mostly performed in
cell culture experiments and using animal models (2). Little is known
about the transcriptional changes induced in vivo by insulin
in human tissues, particularly in skeletal muscle, the main site of
insulin-dependent glucose disposal and the major site of
insulin resistance in type 2 diabetes mellitus. Although it has been
demonstrated that insulin can modulate the mRNA levels of some
important genes in human skeletal muscle, such as hexokinase II, Glut4,
p85 It is likely that insulin coordinates a complex program of
transcriptional changes in target tissues, which represents the molecular basis of its action. Hence, the characterization of this
global pattern of modifications in skeletal muscle is an important step
for a better understanding of the mechanism of action of insulin and of
its defects in situations of insulin resistance. The development of
microarray technology offers powerful tools for characterizing such
large scale changes in transcript levels. Recently, this methodology
was applied to investigate the effects of intensive insulin treatment
for 10 days on the mRNA profile in skeletal muscle of type 2 diabetic patients (7). Using Affymetrix oligonucleotide microarrays
allowing the analysis of 6800 mRNAs, the expression of about 100 genes was found to be modified during insulin therapy (7). With a
similar methodology, it was also demonstrated that 3 days of insulin
treatment induced changes in the abundance of about 100 mRNAs in
streptozotocin diabetic mice, suggesting a coordinated regulation of
gene expression by insulin in skeletal muscle (8). However, it was
difficult to conclude on the direct role of insulin on gene expression
in these two studies because the observed modifications could be the
consequences of the metabolic changes that occurred during the
treatment with insulin.
To determine the global transcriptional modifications directly produced
by insulin in human skeletal muscle, we used cDNA microarrays (9)
to analyze the changes in the mRNA levels of 29,308 genes and
expressed sequence tags
(ESTs)1 induced by 3 h
of euglycemic hyperinsulinemic clamp in the vastus lateralis muscle of
healthy lean subjects. It is well accepted that the hyperinsulinemic
clamp method allows the individual effect of insulin to be studied, and
the short duration of the insulin infusion was chosen to limit possible
secondary effects due to metabolic modifications. Rapid changes in the
mRNA levels of candidate genes have been previously reported in
human skeletal muscle during similar euglycemic hyperinsulinemic clamp
conditions (5, 6). We report here that insulin directly modulates the
mRNA levels of about 800 genes in human muscle. Most of them are
novel targets of the hormone and belong to functional classes that can
account for most of the biological and metabolic effects of insulin in human skeletal muscle.
Subjects--
Twelve lean healthy Caucasian volunteers (4 men
and 9 women) with a mean (±S.E.) age of 32 ± 4 years and a body
mass index of 22.2 ± 0.6 kg/m2 participated in the
study. None had a familial or personal history of diabetes, obesity,
dyslipidemia, or hypertension, and they were not taking medication
except for oral contraceptives. They were on their usual diet before
the study, and none was engaged in heavy exercise. All subjects gave
written consent after being informed of the nature, purpose, and
possible risks of the study. The experimental protocol was approved by
the ethics committee of Hospices Civils de Lyon (France).
Study Design--
All studies were performed in the
postabsorptive state after an overnight fast. The volunteers were
submitted to a 3-h euglycemic hyperinsulinemic clamp with an insulin
infusion rate of 2 milliunits·min Total RNA Preparation and Amplification--
Frozen tissue
samples were crushed in liquid nitrogen, and total RNA was extracted
with the guanidinium thiocyanate method (5). RNA concentrations and
integrity were assessed using an Agilent 2100 Bioanalyzer (Agilent
Technologies, Massy, France). The mean yield of total RNA was 0.28 ± 2 µg/mg of muscle (wet weight) and was not different for the
biopsies taken before and after the clamp.
RNA preparations from 6 subjects (2 men, 4 women) were amplified using
the MessageAmp antisense RNA kit (Ambion, Austin, TX) in order to
generate probe for hybridization to the cDNA microarrays. Briefly,
the procedure consists of reverse transcription of 1 µg of total RNA
with a oligo(dT) primer bearing a T7 promoter sequence followed by
in vitro transcription of the resulting DNA with T7 RNA
polymerase to generate antisense RNA copies of each mRNA (10). This
amplification procedure, now largely accepted, has been validated
before, and it has been demonstrated that it does not distort the
relative abundance of individual mRNAs within an RNA population
(10, 11).
Probe Labeling and Hybridization--
Ten µg of
antisense RNA from basal and insulin-treated conditions of each of the
six subjects were labeled with cyanine (Cy) 3 or Cy5 dyes during a
random-primed reverse transcription reaction using the CyScribe
First-Strand cDNA labeling kit (Amersham Biosciences). They were
hybridized overnight at 65 °C to the cDNA microarray according
to the protocol recommended by the Stanford Functional Genomics
Facility (www.microarray.org/sfgf/jsp/home.jsp). The cDNA
microarrays consisted of PCR-amplified cDNAs printed on glass slides with 42,557 spots representing 29,308 UniGene clusters.
Analysis of Microarray Data--
The six slides were scanned
with a GenePix 4000A microarray scanner (Axon Instruments, Union City,
CA), and the images were analyzed using Genepix pro 3 software. Data
files were entered into the Stanford Microarray Data base
(genome-www5.stanford.edu/MicroArray/SMD). A uniform scale factor was
applied to normalized signal intensities between Cy5 and Cy3. Flagged
spots and spots with an average intensity below 2.5-fold above the
background were not retained for further analysis. The
log2(Cy5/Cy3) ratio of the other spots was calculated for
each slide. To compare the results from the different subjects, data
from each slide were normalized in log space to have a mean of 0 and a
S.D. of 1 by using the Cluster program (46). Only cDNAs with
recorded data on the six slides were then selected for further
analysis. At this stage, 16,140 spots were recovered. Genes with
significant changes in mRNA levels in response to insulin were
identified using the Significant Analysis of Microarrays (SAM)
procedure (12), a validated statistical technique for identifying
differentially expressed genes across high density microarrays. This
procedure provides a list of "significant" genes and an estimate of
the false discovery rate, which represents the percentage of genes that
could be identified by chance (9, 12).
Quantitation of mRNAs Using Real-time
RT-PCR--
First-strand cDNAs were first synthesized from 500 ng
of total RNA in the presence of 100 units of Superscript II
(Invitrogen) using both random hexamers and oligo(dT) primers (Promega,
Charbonnières, France). The real-time PCR was performed using a
LightCycler (Roche Diagnostics) in a final volume of 20 µl containing
5 µl of a 60-fold dilution of the RT reaction and 15 µl of reaction
buffer from the FastStart DNA Master SYBR Green kit (Roche Diagnostics)
with 3 mM MgCl2 and the specific forward and
reverse primers (Eurogentec, Seraing, Belgium). The list of the primers
is available in Table I. After
amplification, a melting curve analysis was performed to verify the
specificity of the reaction. For quantification, a standard curve was
systematically generated with six different amounts (150-30,000
molecules/tube) of purified target cDNA cloned in the pGEM plasmid
(Promega). The analysis was performed using the LightCycler software
(Roche Diagnostics).
Euglycemic Hyperinsulinemic Clamp--
To investigate the effect
of insulin on gene expression, healthy lean volunteers were submitted
to a 3-h euglycemic hyperinsulinemic clamp to achieve
supra-physiological plasma insulin concentrations (5, 6). Insulinemia
increased from 41 ± 3 pM in the basal state to
858 ± 52 pM during the last hour of the clamp. Plasma free fatty acid concentration decreased from 463 ± 64 to 34 ± 4 mM. The glycemia was clamped at 4.6 ± 0.1 mM. The rate of glucose infusion required to maintain
euglycemia during the last hour of the clamp was 11.1 ± 0.6 mg·kg Analysis of cDNA Microarray Data--
For cost issue, the
cDNA microarray experiments were performed with the RNA
preparations from 6 subjects (2 men and 4 women) of the 12 involved in
the study. Their characteristics and their metabolic responses during
the clamp did not differ from the data of the whole group (data not
shown). The cDNA microarrays used in the present work allowed
analysis of the changes in mRNA levels of 29,308 genes and ESTs.
The SAM procedure (9, 12) was performed on 16,140 spots for which
signals were recovered in the six experiments. With an estimated false
discovery rate of 4%, we found 1,065 cDNAs that were
differentially regulated by insulin. As shown in the supplemental
table, this procedure selected cDNAs for which the log2(Cy5/Cy3) ratio changed in the same direction on the
six slides. This indicated that the corresponding genes were modulated
by insulin in the same way in the six subjects. Because of replications of some cDNAs on the microarray, the 1065 cDNAs corresponded to 762 different genes (a list is available in the supplemental table). Of
these 762 genes, 478 were up-regulated, and 284 were down-regulated during the 3 h of the hyperinsulinemic clamp. Fig.
1 shows that the majority
(n = 322) of the up-regulated genes displayed a more than 2-fold change in mRNA levels, as estimated by the mean Cy5 on
Cy3 intensity ratio of the 6 experiments. Only 13 increased 4-fold or
more. Among the down-regulated genes, 104 displayed a 2-fold or more
decrease in expression level.
To test the validity of the microarray experiments and the SAM
procedure, the changes in mRNA levels of 9 genes were quantified using real-time RT-PCR in muscles of the whole group of subjects who
underwent the 3-h hyperinsulinemic clamp. These genes displayed wide
differences in their response to insulin on the microarray (from
We further verified that the procedures used in the microarray
experiment did not select only genes with high expression in muscle.
Using the Stanford Online Universal Resource for Clones and ESTs
(SOURCE at source.stanford.edu) and the published reconstruction of the
human skeletal muscle transcriptional profile (13-15), we found that
32 of the 762 insulin-regulated genes have been reported to be more
expressed in the muscle than in other tissues, with only 6 belonging to
the 400 genes considered as highly expressed in skeletal muscle (13,
15). Conversely, about 150 genes, mostly ESTs with unknown functions,
had never been found to be expressed in the skeletal muscle. This
suggested that the procedures used in the study did not select genes
according to their mRNA expression levels and, thus, allowed to
analyze the effect of insulin on the global muscle transcriptome.
However, because the analysis was performed in tissue biopsies, it
could not be exclude that regulation of gene expression in other cell
types than in muscle cells might have contributed to the detected changes.
Of the 762 genes that showed a regulation during the hyperinsulinemic
clamp, 627 have a known chromosomal location in the data bases (see the
supplemental table). No gene was found on chromosome Y due to the fact
that women participated in the study and the procedure to select the
regulated genes excluded those that did not give a signal in the six
experiments. Analysis of the chromosomal location revealed that the
insulin-regulated genes are widely distributed among the chromosomes
and the number of genes on each chromosome is grossly proportional to
the length of the chromosome (e.g. 56 genes located on
chromosome 1 and 10 on chromosome 21). It could be noted that 6 regions
(5q31, 6p21.3, 12q24, 16p13.3, 17p13.1, and 22q13.2) are characterized
by the co-localization of five or more regulated genes. None of these loci has been reported to be linked with insulin resistance or type 2 diabetes in genetic studies. However, four of these loci (5q31, 6p21.3,
12q24, 17p13.1) have been linked to physical performance and
health-related fitness phenotypes (16).
Functional Classification of the Regulated Genes--
Gene
ontology annotations, SOURCE and PubMed (www.ncbi.nlm.nih.gov) were
used to assign the regulated genes into functional categories (see the
supplemental table for a complete list of the genes and their
classification). Of the 762 genes found to be regulated by insulin, 353 (163 up and 190 down) corresponded to ESTs or hypothetical proteins.
Among them, 16.5% displayed a more than 2.5-fold increased expression
during the clamp and 20.2% displayed a more than 2-fold decreased,
suggesting that a number of new insulin-regulated genes will probably
emerge from the completion of the genome annotation. Of the 409 genes
with known functions, the majority corresponded to genes coding for proteins involved either in the transcriptional and translational regulation (29%), intermediary and energy metabolisms (14%), or in
intracellular signaling (12%). Other functional categories consisted
of genes involved in cytoskeleton and vesicle traffic (9%), receptors,
carriers, and transporters (8%), components of the
ubiquitin/proteasome pathways (7%), and
the immune response (5.5%). In addition, 63 genes (42 up, 21 down) did
not correspond to one of these major categories. Figs. 2 and
3 are schematic representations of these
main metabolic and functional pathways with indications of the number
of up- and down-regulated genes. The different functional groups are
discussed in more details below.
Transcriptional and Translational Regulation--
One of the most
remarkable biological effects of insulin is its profound impact on the
turnover of cellular proteins mainly through an increase of gene
transcription and a stimulation of mRNA translation (2). It is,
thus, not surprising that the majority of the genes found to be
regulated during an acute insulin infusion codes for transcription
factors (such as basic transcription factor 3, general transcription
factor 2A, polymerase II transcription cofactor 4) and for proteins
involved in RNA transport, processing, and translation (splicing
factors 1 and 10, karyopherins 1 and 3, eukaryotic translation
initiation factors 2 and 3, eukaryotic translation elongation factor
1). In addition, the mRNA expression of 14 ribosomal proteins,
including 4 of the mitochondria, is markedly induced during the
hyperinsulinemic clamp. Insulin also affects the expression of 20 genes
coding for chaperones, heat-shock proteins, or enzymes involved in
post-translational modifications, hence contributing to the complete
process of protein synthesis that generally includes proper protein
folding and a number of post-translational modifications.
Intermediary and Energy Metabolism--
In skeletal muscle,
insulin increases the uptake of glucose, fatty acids, and amino acids
and directs their metabolic fates into specific pathways. The storage
of glucose into glycogen and fatty acid into triglycerides is enhanced
by insulin, whereas amino acids are mostly used for protein synthesis.
Insulin also increases energy metabolism and ATP synthesis mainly
by stimulating glucose oxidation, whereas fatty acid oxidation is
almost completely inhibited (17). The observed changes in the gene
expression pattern during the hyperinsulinemic clamp are in agreement
with these metabolic effects.
Hexokinase II catalyzes the phosphorylation of glucose into glucose
6-phosphate after its entry into the muscle cells. Changes in the level
and the activity of this key enzyme of glucose metabolism would
markedly affect both the glycolytic and the glyconeogenic fluxes.
Up-regulation of hexokinase II expression by insulin has been
previously reported in human muscle (3, 6). In addition to hexokinase
II, the increased expression of glycogenin and the catalytic subunit of
protein phosphatase 1 (PPP1G) is in agreement with the stimulation by
insulin of glycogen synthesis. Glycogenin is a self-glucosylating
protein involved in the initiation of glycogen synthesis (18), and
protein phosphatase 1G is one of the regulatory enzymes controlling
glycogen synthase activity (19).
As for glucose, insulin increases fatty acid uptake and
triglyceride storage in skeletal muscle (20). It has been recently demonstrated that part of the mechanism of this effect involved the
translocation of the fatty acid transporter FAT/CD36 to the plasma
membrane in muscle cells (21). Both the microarray and the quantitative
RT-PCR data provide evidence that insulin increases FAT/CD36 and fatty
acid CoA ligase (FACL2) expression in human skeletal muscle. In
addition, the mRNA expression of the adipose differentiation-related protein (ADRP, also called adipophilin) is
increased during the hyperinsulinemic clamp. ADRP is a lipid droplet-associated protein involved in the package of neutral lipids in
most mammalian cells (22). All these effects may, therefore, contribute
to higher rates of entry, esterification, and storage of long chain
fatty acids in muscle cells in response to insulin stimulation.
Insulin increases oxygen consumption and ATP synthesis in most cells.
The generation of ATP and the coupling between oxidation and
phosphorylation mostly rely on the activity of the electron transport
chain in the inner mitochondrial membrane. We found that the mRNA
levels of seven proteins from the different enzymatic complexes of the
electron transport chain were increased by insulin. Interestingly, it
was recently reported that the expression of a number of these proteins
is reduced in a streptozotocin diabetic rats and that the treatment
with insulin leads to correction of this defect (8). All these data
thus support a direct and an important role of insulin in the control
of the mitochondrial electron transport chain at the transcriptional level.
Intracellular Signaling--
Insulin binding to its membrane
receptor triggers the activation of inter-related signaling cascades
mostly through protein-protein interactions and
phosphorylation/dephosphorylation mechanisms (1, 23). However, little
is known regarding the transcriptional regulation of the key proteins
of this signaling network in response to insulin. We found previously
that the mRNA levels of the p85 Cytoskeleton and Vesicle Traffic--
The mRNA levels of 19 genes encoding for proteins involved in the vesicle traffic and 17 genes for cytoskeleton proteins were changed by the insulin treatment.
In muscle cells in culture, insulin induces cytoskeleton reorganization
associated with membrane ruffling and localized accumulation of
pinocytotic/endocytotic vesicles (27). It is also well described that
one of the major actions of insulin is to promote glucose uptake in
skeletal muscle through the translocation of the Glut4 facilitative
glucose transporter from an intracellular vesicle pool to the plasma
membrane (28). This effect is dependent upon both microtubule- and
actin-based cytoskeletal structures (29). The microtubule network and
the cytoskeleton are also implicated in the proper subcellular
localization of signaling molecule, such as the
IRS-1-phosphatidylinositol 3-kinase complex (27). We found that several
important components of the actin network are target genes of insulin
in vivo in human skeletal muscle. The mRNA levels of
actin-b, cofilin 2, vinculin, and supervilin among others are markedly
increased after 3 h of insulin infusion. Furthermore, insulin also
regulated the mRNA levels of genes involved in the microtubule
network like the microtubule-based motors dynein and kinesin (PIN,
KIF5B, KIF1C, KIFAP3). In addition to actin- and microtubule-based
cytoskeletal structures, the translocation of Glut4 requires a number
of proteins involved in the docking and fusion process of the vesicles
with the plasma membrane. This includes members of the v-SNARE
and t-SNARE complexes and small GTP-binding proteins involved in
regulating membrane traffic. Among them, we found that insulin
regulates the expression of the GTP binding Rab5 that has been recently
shown to interact with the motor protein dynein and to participate in
Glut4 internalization (30).
Receptors, Carriers, and Transporters--
Insulin modulates the
mRNA levels of 33 genes coding membrane receptors and cellular
transporters and carriers. Among them, we found that insulin
up-regulates Glut8 (also called GlutX1 or SLC2A8), a recently
discovered novel glucose transporter (31) that has been shown to be
involved in insulin-stimulated glucose uptake in different cell models
(31, 32). Previous studies demonstrate that the expression of Glut4 is
induced in human skeletal muscle during an hyperinsulinemic clamp
(4-6). Unfortunately, this could not be shown in the present study
because the cDNA probe corresponding to Glut4 (SLC2A4) was not
spotted on the microarrays. Among the membrane receptors found
to be modulated by insulin during the hyperinsulinemic clamp, four
belong to the cytokine receptor family, suggesting an effect of insulin
on the expression of genes involved in the immune response.
Immune Response and Cytokine Actions--
During the last decade,
growing number of evidence was accumulated, demonstrating that
cross-talks occur between insulin and cytokine signaling pathways (33).
Moreover, diseases with insulin resistance (e.g. type 2 diabetes, obesity) are often associated with increased levels of
inflammation markers (34). We found that hyperinsulinemia induces
significant changes in the expression levels of 24 genes involved in
cytokine action and immune response in human skeletal muscle, including
interleukin 17D, transforming growth factor Ubiquitin-Proteasome Pathway--
It is currently recognized that
insulin promotes net skeletal muscle protein synthesis and that
inhibition of protein breakdown plays an important role in this process
(35). Rather unexpectedly, we found that insulin increases the
expression level of 17 mRNAs of the ubiquitin-conjugating enzymes
and 11 mRNAs of the proteasome components. This may suggest
increased degradation of peculiar proteins after insulin infusion in
human skeletal muscle. Indeed, its is well documented that controlled
degradation of specific proteins by the ubiquitin-proteasome system
plays an important role in the execution of various biological events,
such as the regulation of different signal transduction pathways,
including insulin action. Indeed, the proteasome has been directly
involved in the selective degradation of the insulin receptor
substrates IRS-1 and -2 (36) during prolonged insulin
stimulation. Moreover, direct interaction of the glucose transporters
Glut1 and Glut4 with members of the ubiquitin family has been evidenced
and proposed to play a role in the control of glucose uptake (37).
The ubiquitin-proteasome system is also involved in the regulation of
transcription (38). We found that insulin increases the mRNA of
USP16, which participates in the deubiquitination of histones H2A and
H2B (39), a process that has been correlated with transcriptionally
active DNA. This is consistent with the general activation of gene
transcription upon insulin stimulation. In addition, the
ubiquitin-proteasome pathway has been implicated in the control of the
amount of specific transcription factors such as nuclear factor Summary and Conclusion--
The present data demonstrate that
insulin infusion for 3 h during a hyperinsulinemic euglycemic
clamp results in profound changes in the mRNA levels of about 800 genes in human skeletal muscle. About half of them are ESTs with still
unknown functions. The others could be classified into functional
categories that can support most of the biological and metabolic
effects of insulin. The microarray data were confirmed by measuring the
mRNA levels of a subset of genes using real-time RT-PCR. Moreover,
some of these genes, such as hexokinase II and p85
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
phosphatidylinositol 3-kinase, or lipoprotein lipase (3-5),
most of these studies were focused on a small number of selected genes
involved in glucose and lipid metabolism (5, 6) and did not give a
global view of insulin action on gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1·kg
1,
as described previously (5). Percutaneous biopsy of the vastus lateralis muscle was obtained with a Weil Blakesley plier before and
after 3 h of insulin infusion (5). Average muscle samples of
63 ± 4 mg (wet weight) were obtained, immediately frozen in liquid nitrogen, and stored at
80 °C until total RNA preparation.
Sequences of the primers used for mRNA quantitation by real-time
RT-PCR
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1·min
1, indicating a normal effect
of insulin on whole body glucose disposal rate (5, 6). Glucose
oxidation rate, as determined by indirect calorimetry, rose from
1.3 ± 0.2 mg·kg
1·min
1 before
insulin infusion to 3.5 ± 0.2 mg·kg
1·min
1 at the end of the clamp
(p < 0.001). Glucose storage, estimated from the
non-oxidative glucose disposal rate, was 7.6 ± 0.6 mg·kg
1·min
1 during the last hour of
insulin infusion. Skeletal muscle biopsies were taken before and at the
end of the hyperinsulinemic clamp, and total RNA was prepared from each
tissue samples.
View larger version (10K):
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Fig. 1.
Distribution of the -fold changes in
mRNA levels induced by insulin in human skeletal
muscle.
1.95
to 5.04-fold change) and belong to the 3 main functional categories
that were defined to classify the insulin-regulated genes (see
"Functional Classification of the Regulated Genes"). As
shown in Table II, the changes measured
by real-time RT-PCR confirmed the microarray data for all genes except
one. The down-regulation of the nuclear receptor NR5A2 observed by
microarray was not reproduced by RT-PCR (Table II). However, it should
be mentioned that the absolute mRNA level of NR5A2 (about 0.05 amol·µg
1 total RNA) was closed to the lower limit
of the RT-PCR assay.
Comparison of microarray results with quantitative RT-PCR
View larger version (33K):
[in a new window]
Fig. 2.
Schematic representations of the effects of
insulin on gene expression in skeletal muscle. The main functional
categories of the regulated mRNAs are shown. The number of
up-regulated genes is indicated with white boxes, and the
number of down-regulated genes is indicated with gray
boxes.
View larger version (30K):
[in a new window]
Fig. 3.
Schematic representations of the effects of
insulin on gene expression in skeletal muscle. The main functional
categories of the regulated mRNAs are shown. The number of
up-regulated genes is indicated with white boxes, and the
number of down-regulated genes is indicated with gray
boxes.
regulatory subunit of
phosphatidylinositol 3-kinase is increased during a hyperinsulinemic
clamp in healthy volunteers (5). Using cDNA microarray, we
confirmed this observation, and more importantly, we identified at
least 49 other insulin-target genes coding for proteins potentially
involved in signaling. Not surprising with respect to the important
role of protein phosphatases both in insulin action and in insulin
signaling regulation (24), 12 of these 49 genes code for phosphatases.
In addition, insulin induces the mRNAs of 2 phosphodiesterases
(PDE4D and PDE7A) and of the protein kinase A anchor protein 6 (AKAP6),
that may participate in the counter-regulatory effect of insulin on the
cAMP pathway. This well described anti-cAMP effect of insulin is
corroborated by the decreased expression of CREBBP mRNA, a
cofactor that participates in the transcriptional regulation of the
cAMP-responsive genes (25). Importantly, we also found that insulin
strongly increases the mRNA level of CAP, the CBL-associated
protein (SH3D5). The adaptor protein CAP is involved in the interaction
of CBL with insulin receptor, and it has been demonstrated that the
CAP·CBL complex dissociates from the insulin receptor and moves to
lipid raft upon insulin stimulation, generating a pathway that is
crucial in the regulation of glucose transport (26).
2, interleukin 13 receptor, and interferon
receptor 2. Some of these genes may
represent novel candidates for the link between insulin action and
inflammation (34).
B1,
retinoid X receptors
, peroxisome proliferator-activated receptors
and
, thyroid hormone receptor, or sterol regulatory
element-binding proteins (38, 40-43). Most of these transcription
factors have been directly implicated in or related to insulin action
(44, 45). All together, these data and our observation of a marked
impact of insulin on the expression of members of the
ubiquitin-proteasome system strongly suggest that this pathway may play
an important role in insulin action in human muscle.
phosphatidylinositol 3-kinase, have been previously reported to be
regulated by insulin in separated studies. Therefore, our data markedly
increased the list of the 120-150 target genes of insulin that was
previously established, mostly on the basis of experiments with animal
models. Among them, the ubiquitin-proteasome pathway emerged as an
important component of insulin action in human muscle. A prominent
issue now is to understand how insulin orchestrates the coordinated regulation of all these target genes. The mechanism of action of
insulin on gene expression in skeletal muscle is still largely unknown.
The identification of common insulin response elements in the promoter
sequences of group of genes will help the discovery of the
transcription factors linking the effect of insulin on multiple genes
simultaneously. In addition, because accumulating data indicate that
defects in basal and in insulin-regulated gene expression may be
involved in the etiology of insulin resistance and type 2 diabetes
mellitus (2, 6), certain of the 800 genes identified in the present
work are potential novel candidates in pathologies with altered insulin responsiveness.
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ACKNOWLEDGEMENTS |
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We thank C. Urbain, J. Peyrat, and V. Peloux for excellent technical assistance. We thank D. Langin for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by INSERM Action Thématique concertée Nutrition Grant 4NU10G and by grants from the Institut de Recherche Servier, Région Rhône-Alpes, and Claude Bernard Fondation.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.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental table.
§ Chargée de Recherche from Institut National de la Recherche Agronomique. To whom correspondence should be addressed: INSERM U.449, Faculté de Médecine R Laennec, Rue G. Paradin, F-69372 Lyon Cédex 08, France. Tel.: 33-478-77-86-29; Fax: 33-478-77-87-62; E-mail: srome@univ-lyon1.fr.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300293200
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ABBREVIATIONS |
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The abbreviations used are: EST, expressed sequence tag; RT, reverse transcription; CREBBP, cAMP-response element-binding protein (CREB)-binding protein; SNARE, soluble N-ethylmaleimide factor attachment protein receptor; IRS, insulin receptor substrate.
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