J Biol Chem, Vol. 274, Issue 33, 23078-23084, August 13, 1999
Altered Gene Expression Pattern in the Fatty Liver Dystrophy
Mouse Reveals Impaired Insulin-mediated Cytoskeleton Dynamics*
Martin
Klingenspor
§,
Ping
Xu,
Robert D.
Cohen,
Carrie
Welch¶
, and
Karen
Reue**
From the Department of Medicine, University of
California, and The Lipid Research Laboratory, West Los Angeles
Veterans Affairs Medical Center, Los Angeles, California 90073 and the
¶ Department of Pathology, University of California,
Los Angeles, California 90045
 |
ABSTRACT |
The mouse fatty liver dystrophy (fld)
mutation is characterized by transient hypertriglyceridemia and fatty
liver during the neonatal period, followed by development of a
peripheral neuropathy. To uncover the metabolic pathway that is
disrupted by the fld mutation, we analyzed the altered
pattern of gene expression in the fatty liver of fld
neonates by representational difference analysis of cDNA.
Differentially expressed genes detected include a novel member of the
Ras superfamily of small GTP-binding proteins, a novel Ser/Thr kinase,
and several actin cytoskeleton-associated proteins including actin,
profilin, 
-actinin, and myosin light chain. Because these proteins
have a potential functional link in the propagation of hormone signals,
we investigated cytoskeleton dynamics in fld cells in
response to hormone treatment. These studies revealed that
preadipocytes from fld mice exhibit impaired formation of
actin membrane ruffles in response to insulin treatment. These findings
suggest that the altered mRNA expression levels detected in
fld tissue represent a compensatory response for the nonfunctional fld gene and that the fld gene
product may be required for development of normal insulin response.
| |
INTRODUCTION |
Fatty liver dystrophy
(fld)1 is a
recessive mutation that arose spontaneously in an inbred mouse strain
and is named for the hallmark fatty liver present in neonatal mice and
impaired nerve function apparent in adult animals (1). The fatty liver
develops as the newborn mice begin to suckle milk and is accompanied by elevated plasma triglyceride levels (1000 mg/dl). However, the fatty
liver and hypertriglyceridemia resolve spontaneously when mice reach
2-3 weeks of age, at which time the mutant animals begin to develop a
peripheral neuropathy that persists throughout their lifetime. The
neuropathy is associated with abnormal myelin formation and axonal
degeneration in the peripheral nerve (2). The fld gene has
been mapped to mouse chromosome (Chr) 12 (3), but neither the mutant
gene nor the biochemical basis for the lipid and nerve defects have
been identified (reviewed in Ref. 4).
Previous studies of the fld fatty liver have revealed
altered mRNA levels for proteins involved in lipid metabolism,
including hepatic lipase (60% reduction), apolipoprotein A-IV
(100-fold induction), and apolipoprotein C-II (6-fold induction) (1). Using quantitative two-dimensional gel electrophoresis, we have detected approximately 25 additional proteins that show significantly altered levels in the fatty liver of neonatal fld mice; the
majority of these proteins could not be identified on the basis of
current two-dimensional protein data base information (5). The goal of
the present study was to identify genes with altered expression levels
in the fatty liver of fld mice, with the intent of
uncovering a specific metabolic pathway affected by the mutation.
Toward this end, we have employed representational difference analysis (RDA) to isolate cDNA tags corresponding to mRNAs with altered expression in the fld fatty liver.
The RDA technique was originally developed and applied to isolate
differences between complex genomes, such as genetic lesions in tumors
that result from DNA deletion, insertion, or rearrangement (6, 7).
Subsequently, RDA has been adapted for use at the cDNA level and
employed to isolate sequence tags corresponding to mRNA species
that are differentially expressed between two cell populations (8-11).
In cDNA-RDA, representations generated by PCR from two cDNA
populations of interest are compared in successive rounds of
subtraction-hybridization, kinetic enrichment, and selective amplification, resulting in sequences corresponding to mRNA species that are expressed at different levels in the two populations.
Using RDA we have isolated 22 mRNA species with altered expression
levels in the fatty liver of fld neonates compared with their wild type counterparts. These RDA sequences include several that
encode proteins associated with the actin cytoskeleton, a putative
novel Ser/Thr protein kinase, and a putative novel member of the Ras
superfamily of small GTP-binding proteins. Because members of the Ras
and kinase protein families are functionally linked to components of
the actin cytoskeleton in the process of hormone signal propagation, we
investigated whether hormone-induced changes in the cytoskeleton were
impaired in fld cells. Indeed, it was found that
preadipocytes isolated from fld mice fail to form actin
membrane ruffles in response to insulin. These results establish that
fld mouse tissues exhibit altered expression levels of
cytoskeleton-associated and putative signal transduction proteins, which is associated with impaired cytoskeleton response to hormones such as insulin.
| |
EXPERIMENTAL PROCEDURES |
Animals
Nontested breeding pairs of the mouse strain
BALB/cByJ-fld were obtained from the Mouse Mutant Resource
Colony at the Jackson Laboratory (Bar Harbor, ME) and bred to produce
fld/fld offspring used in these studies. The
fld/fld pups were recognized at 3-5 days of age by their
smaller body size, swollen abdomen, pale liver, and delayed onset of
fur growth. Adult fld/fld mice were recognized by the
characteristic whole body tremor and clenching of hind legs when
handled. Throughout the text, the term "fld" is used to
indicate mice of the fld/fld genotype, and "wild type" is used to indicate mice of genotypes +/+ and
+/fld, which both appear phenotypically normal.
Representational Difference Analysis
Representational difference analysis was performed as described
below, based on the protocol by Hubank and Schatz (11).
Amplicon Generation--
RNA was prepared from liver of wild
type and fld littermates at 6 days of age by the acid
phenol-guanidine method (Trizol; Life Technologies, Inc.). Total RNA
(400-500 µg) obtained from two isogenic littermates was combined for
poly(A)+ RNA isolation (Poly(A)-Tract IV, Promega, Madison, WI), and 5 µg of the resulting poly(A)+ RNA was used for cDNA synthesis
(Superscript Choice System, Life Technologies, Inc.). 1 µg of
cDNA was digested with DpnII (New England Biolabs,
Beverly, MA), extracted with phenol-chloroform, and precipitated in
ethanol. cDNA fragments were ligated to R-Bgl24 oligomers in the
presence of R-Bgl12 oligomers (Operon Technologies, Alameda, CA; see
Ref. 11 for sequences of oligonucleotides used in RDA). Ligated
cDNA fragments were diluted and amplified for 19 cycles with
R-Bgl24 as a primer. The resulting amplicon preparation was extracted
with phenol-chloroform, and R-Bgl24 adaptors were removed by
DpnII digestion. All manipulations were performed in parallel for wild type and fld samples.
Tester Preparation--
Amplicons prepared from wild type and
fld RNA were each used as tester in independent reactions.
For tester preparation, 10-20 µg of amplicon was subjected to gel
electrophoresis in 1.2% low melting agarose. The size range of
100-1500 base pairs was excised and gel purified using Qiaex II resin
(Qiagen, Valencia, CA). 1 µg of gel purified tester was ligated to
the new oligomer, J-Bgl24. To permit precise adjustment of
driver/tester ratios in subtractive hybridization steps, DNA
concentrations were measured fluorometrically using Hoechst dye and
calf thymus DNA as a standard (12). The accuracy of DNA quantitation
was verified on agarose gels.
Subtractive Hybridization--
In the first round of subtractive
hybridization, 40 µg of driver and 0.4 µg of tester were mixed,
extracted with phenol-chloroform, and precipitated with ethanol. The
resulting pellet was washed twice in 70% ethanol, dried, and
thoroughly dissolved in 4 µl of hybridization buffer (30 mM EPPS, pH 8.0, 3 mM Na2EDTA). The DNA droplet was overlaid with mineral oil and denatured at 98 °C for
5 min in a PCR machine. The reaction tube was immediately transferred
to a 67 °C water bath, and 1 µl 5 M NaCl was added. The tester-driver mixture was incubated at 67 °C for 20 h.
Amplification of Difference Products--
After hybridization
for 20 h, the hybridization mixture was diluted step-wise in TE
(10 mM Tris-Cl, 1 mM EDTA, pH 8.0) in the
presence of 40 µg of yeast tRNA to a final volume of 400 µl. The
diluted hybridization mix was subjected to 10 cycles of PCR amplification using J-Bgl24 as a primer. The resulting PCR product was
extracted with phenol-choroform, precipitated with ethanol in the
presence of glycogen, and treated with Mung Bean nuclease (New England
Biolabs, Beverly, MA) for 30 min at 30 °C to remove single-stranded
DNA, which consisted primarily of unhybridized driver sequences. The
Mung Bean nuclease reaction mixture was denatured at 95 °C for 8 min
and chilled on ice. The first difference product was generated from the
denatured Mung Bean nuclease mixture by 19 cycles of PCR amplification
using J-Bgl24 as a primer. In total, three or four rounds of
subtractive hybridization and selective amplification of tester-tester
hybrids were performed. In each new round of subtraction the difference
product of the previous subtraction was used as the tester in
combination with a fresh aliquot of the initial amplicon from the
opposite source as driver (40 µg). Two RDA experiments were performed
with different tester/driver ratios: in one experiment, tester/driver
ratios were 1:100, 1:400, 1:80,000, and 1:800,000 for rounds 1-4 (11);
the second experiment used a constant ratio of 1:100 for a total of
three rounds. Difference products resulting from the final round of
subtraction from each experiment were cloned into pBlueScript SK+
(Stratagene, La Jolla, CA).
Northern Blot and DNA Sequence Analysis of RDA Clones
Northern blot analysis was performed using 10 µg of total RNA
from liver of 6-day-old and 3-month-old wild type and fld
mice as described (13). RDA plasmid inserts were sequenced by the dideoxy method, and sequences were compared with those in nucleotide data bases using the BLAST algorithm (14).
Reverse Transcriptase-PCR
Total RNA from liver, adipose, and sciatic nerve was treated
with DNase I (Ambion, Austin, TX) at 37 °C for 30 min to remove any
potential contaminating genomic DNA. cDNA was synthesized from 2 µg of DNase-treated RNA using avian myeloblastosis virus reverse
transcriptase and oligo(dT) primer (cDNA Cycle Kit, Invitrogen, Carlsbad, CA). PCR amplification was performed using one-tenth of the
resulting cDNA in a total volume of 50 µl containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl2, 0.001% gelatin, 200 µM
dNTPs, 0.01 µg/µl forward and reverse primers, and 1 unit of AmpliTaq (Perkin-Elmer, Foster City, CA). PCR cycling conditions consisted of a hot start "touchdown" protocol in which the
annealing temperature was gradually decreased over a 10 °C range
(from 63 to 53 °C) to reduce nonspecific priming (15), and products
were analyzed by agarose gel electrophoresis. The initial reaction conditions were denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min, and extension at 72 °C for 2 min. The annealing temperature was decreased 0.5 °C at each cycle for 20 cycles and maintained at 53 °C for an additional 12 cycles. Products were analyzed by agarose gel electrophoresis. The gene specific primers employed were as follows: Ifld-1, forward, 5'-TCGGGGAACCACTTGATG-3', and reverse, 5'-ACTACGCCCCGACGGTGTTTGA-3'; Ifld-2, forward,
5'-ATGACGCGCTGCGGGACAAG-3', and reverse, 5'-CTGCCAGCCAGCATGGTGAAGAG-3';
HPRT, forward, 5'-CACAGGACTAGAACACCTGC-3', and reverse,
5'-GCTGGTGAAAAGGACCTCT-3'; and PMP-22, forward,
5'-ACACTGCTACTCCTCATCAGTGAG-3', and reverse,
5'-CAGGATCACATAGATGATACCACTG-3'.
Chromosomal Localization of RDA Sequences
Selected RDA sequences were mapped in the mouse genome using a
[(C57BL/6J × Mus spretus)F1 × C57BL/6J]
interspecific backcross (16). Isolated inserts were prepared from RDA
plasmids, radiolabeled, and hybridized to restriction-digested genomic
DNA from C57BL/6J and M. spretus mice to identify
restriction fragment length variants. Distinct variants were then
scored in 67 backcross mice that had been typed for more than 350 genetic markers. Linkage was detected with Map Manager version 2.6.5 (17).
Primary Cell Culture and Actin Cytoskeleton Staining
Preadipocytes were released from inguinal fat pads of 4-week-old
wild type and fld mice by collagenase digestion followed by
filtration through 62-µm nylon mesh (18). Preadipocytes were recovered by centrifugation at 800 × g for 5 min at
room temperature and plated onto sterile coverslips at a density of
4 × 104 cells/coverslip in 6-well culture dishes and
maintained for 24 h in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 15 nM insulin. After 24 h, cells
were approximately 50% confluent.
For hormone stimulation studies, cells were incubated in Dulbecco's
modified Eagle's medium devoid of serum and insulin for 18 h to
bring them to a quiescent state. Cells were subsequently treated for 10 min at 37 °C with phosphate-buffered saline alone, with 1% fetal
calf serum, or with 40 ng/ml insulin and then fixed and stained with
tetramethylrhodamine isothiocyanate-phalloidin (Sigma) as described
(19). Stained cells were observed with a Zeiss Axioskop microscope at
an excitation wavelength of 540 nm and an emission wavelength of 590 nm.
| |
RESULTS |
Isolation of Differentially Expressed Sequence Tags from fld Liver
Using RDA--
RDA was performed using RNA from the liver of
fld and wild type littermates at 6 days of age, at which
time the fld mice exhibit an enlarged fatty liver. Both
possible tester-driver combinations were employed to allow isolation of
sequences expressed at higher abundance in fld compared with
wild type ("F clones"), as well as those expressed at higher
abundance in wild type compared with fld ("W clones").
Following three or four rounds of RDA, the resulting difference
products were cloned into plasmids, and approximately 150 of these were
characterized by sequencing. Based on DNA sequence, clones were
segregated into 22 groups, and a representative of each group was used
to probe Northern blots to confirm
differential expression in liver of
fld and wild type mice. As shown in Fig. 1 and Table
I, these RDA clones correspond to
mRNA species expressed at low, moderate, and high abundance and
include examples that exhibit diminished (i.e. insulin-like
growth factor-binding protein 4, Igfbp4), moderately
increased (i.e. profilin), or vastly increased levels in the
fld fatty liver (i.e. novel sequences
Ifld1, Ifld2, and Ifld3). Some of the
RDA sequences are expressed only at ages during which the fatty liver
phenotype is apparent (i.e. Ifld1, Ifld2,
and Ifld3), whereas others are also expressed in adult mouse
liver (i.e. profilin and Igfbp4).
View larger version (105K):
[in this window]
[in a new window]
|
Fig. 1.
Representative differentially
expressed mRNAs in liver from fld versus
wild type mice. Total RNA was prepared from liver of
6-day-old and 3-month-old wild type (wt) and fld
mice. Replicate membranes containing 10 µg of each RNA sample were
hybridized to RDA clones indicated at left. Hybridization
signals were detected by phosphorimaging after 16-24-h
exposures.
|
|
The F clones could be classified into 16 unique groups based on DNA
sequence alignments, and the W clones could be segregated into 6 groups
(Table I). These sequence groups were evaluated by comparison with
nucleic acid sequence data bases using the BLAST algorithm (14). 14 of
22 of the sequences could be unambiguously identified as known genes
and 3 of 22 were found to be present in the expressed sequence tag data
bases. The remainder either represent novel sequences with no match in
data bases (i.e. F1 and F23) or sequences that are related
but not identical to known genes (i.e. F4, F9, and F24).
Expression levels of the RDA clones were evaluated in RNA isolated from
liver of 6-day-old and adult mice via Northern blot analysis. As
summarized in Table I, 13 of the 16 F clones hybridized to mRNA
species with elevated expression levels in liver from 6-day-old
fld compared with wild type mice; among these was apo A-IV,
which is known to be expressed at dramatically elevated levels in the
fld fatty liver (1). Three of the F clones (F7, F15, and
F20) produced no detectable signal on Northern blots performed with 10 µg of total liver RNA, indicating that they are expressed at low
levels. The W clones corresponding to insulin-like growth
factor-binding protein 4 and troponin C hybridized to mRNA with
reduced levels in the fld liver, whereas four of the W
clones could not be detected on blots containing RNA from 6-day-old
fld and wild type liver. Interestingly, all of the W clones
in this latter group (myosin light chain, myosin heavy chain, troponin I, and -2 actinin) were expressed in the liver of adult
(3-month-old) wild type and fld mice and exhibited elevated
expression levels in adult fld compared with wild type liver
(denoted in Table I by mRNA level measurements enclosed in
parentheses). Of the RDA sequences isolated, only four (one F clone and
three W clones) were confirmed as false positives that were expressed
in wild type and fld liver at similar levels; these
sequences are not included in Table I.
The three cDNA tags that exhibited the greatest level of mRNA
induction in fld compared with wild type liver (Fig. 1)
represented previously undescribed sequences and were given the gene
symbols Ifld1, Ifld2, and Ifld3
(induced in fld-1,
-2, and -3). Ifld1 exhibits 60%
amino acid sequence identity with members of the Ras family of small
guanosine triphosphatases, including conservation of two GTP-binding
sites and an effector domain (Fig.
2a). Ifld2 exhibits
75% amino acid identity with a putative serine/threonine protein
kinase and contains protein kinase C and casein kinase II
phosphorylation recognition motifs (Fig. 2a).
Ifld3 was found to have no significant match to sequences in
current releases of nucleic acid or protein data bases. To determine
whether any of these three novel sequences correspond to the
fld gene on proximal Chr 12, we mapped the genes in a mouse
backcross panel. Ifld1 maps to Chr 5, Ifld2 maps
to Chr 2, and Ifld3 is located on Chr 3 (Fig.
2b). These results suggest that Ifld1 and
Ifld2 represent novel Ras-related and serine/threonine
kinase genes but excludes the three RDA clones as candidates for the
fld gene itself. Additional RDA clones shown in Table I that
were mapped and excluded as fld candidates include the genes
for tricarboxylate transport protein (distal Chr 12, distinct from the
fld locus), profilin (Chr 11), ubiquitin (Chr 11),
androgen-withdrawal apoptosis protein (Chr 5), IgJH DNA with
homology to bcl2 (Chr 4), and insulin-like growth
factor-binding protein 4 (Chr 11).
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 2.
Characterization of novel RDA clones by
sequence analysis and chromosomal localization. a,
deduced amino acid sequence of Ifld1 aligned to members of
the Ras superfamily and Ifld2 aligned to putative Ser/Thr
protein kinases. Identities for alignment of Ifld1
(indicated by black shading) are 59.4% with mouse RhoD
(GenBankTM accession number X84325), 56.4% with mouse Rac2
(also known as En-7, GenBankTM accession number X73247),
56.4% with human RhoA (GenBankTM accession number L25080),
and 59.4% with yeast Rho1 (GenBankTM accession number
M15189). Consensus motifs common to Ras proteins are
underlined: an ATP/GTP-binding site motif A (P-loop)
GXXXXGK(S/T) (35), GTP-binding motif DTAGQ, and a potential
effector region, YXPTVFXXY (36). Identities for alignment of
Ifld2 are 75.8% with dog phosphoprotein
(GenBankTM accession number X99144) and human Ser/Thr
protein kinase (GenBankTM accession number D87119), 41.7%
with ustilago cAMP-dependent protein kinase
(GenBankTM accession number AF025290), and 33.3% with
yeast cAMP-dependent protein kinase (also known as TPK2;
GenBankTM accession number M17073). Consensus motifs common
to these protein kinases are underlined: a casein kinase II
phosphorylation site (S/T)XX(D/E) (37) and a protein kinase
C phosphorylation site (S/T)X(R/K) (38, 39). Numbers at
left refer to the amino acid position in each protein as
given in the data base files referenced. b, chromosomal
localization of Ifld1, Ifld2, and
Ifld3 genes. Mapping was performed using DNA from an
interspecific mouse backcross ((C57BL/6J × M. spretus) × C57BL/6J) Ifld1, Ifld2, and
Ifld3 are indicated on Chrs 5, 2, and 3, respectively;
mapping data have been deposited in the Mouse Genome Data Base under
accession number MGD-JNUM-42314. References for other loci shown can be
obtained from the Mouse Genome Data Base. Chromosomes are drawn to
scale with the ratios of the number of recombinants to the total number
of informative mice and the recombination frequencies ± standard
errors (in centimorgans) indicated to the left.
Numbers in parentheses represent the upper 95%
confidence interval for pairs of loci that cosegregate. The cumulative
distance (cum) of the most distal locus from the centromere
is indicated to the left of the corresponding locus on each
chromosome.
|
|
Although genetic mapping results excluded Ifld1 and
Ifld2 as fld gene candidates, their similarity to
known proteins involved in signal transduction and the striking
induction of the corresponding mRNAs in the fld fatty
liver suggested that they might represent an important secondary
response to the underlying defect and serve as markers for the
metabolic pathway(s) disrupted by the fld mutation. We
therefore examined expression of Ifld1 and Ifld2
mRNA in two other tissues that exhibit abnormal phenotypes in
fld mice, sciatic nerve, and adipose tissue. After
resolution of the fatty liver, fld mice exhibit reduced
myelination and altered protein expression in sciatic nerve and
drastically reduced adipose tissue mass (2, 4). Because of the small
amount of tissue available from nerve and adipose, we used reverse
transcriptase-coupled PCR to examine gene expression in these tissues
from wild type and fld mice. The reverse
transcriptase-coupled PCR results from liver recapitulated those seen
by Northern blot, with vastly increased expression in the liver of
fld neonates (6 days of age), low levels in the liver of
both wild type and fld adult mice (6 weeks of age), and no
detectable products in samples not treated with reverse transcriptase (Fig. 3a). Both
Ifld1 and Ifld2 were also expressed in sciatic nerve, with somewhat lower levels detected in sciatic nerve from adult
fld compared with wild type mice when normalized to levels of HPRT (a ubiquitously expressed mRNA) and PMP22 (a specific marker for peripheral nerve) (Fig. 3b). Ifld1 and
Ifld2 mRNA were detected in inguinal and epididymal
adipose tissue at similar levels in adult wild type and fld
mice. Thus, Ifld1 and Ifld2 are expressed in each
of the three tissues currently known to exhibit phenotypic alterations
in fld mice.
View larger version (62K):
[in this window]
[in a new window]
|
Fig. 3.
Reverse transcriptase-coupled PCR analysis of
Ifld1 and Ifld2 expression in liver,
sciatic nerve, and white adipose tissue of wild type
(wt) and fld mice. Liver RNA was
analyzed in mice at the age of 6 days (neonates) and 6 weeks
(adults); sciatic nerve and adipose RNA were analyzed in
adult mice only and were pooled from 2-4 animals. Gene specific
primers were used to amplify Ifld1, Ifld2, and
control cDNAs (HPRT and PMP22) from reverse transcribed total RNA
(see "Experimental Procedures" for details). As a control, RNA was
directly added to the PCR reaction without reverse transcriptase
treatment ( RT). PCR products shown represent inverted
images of ethidium bromide-stained agarose gels.
|
|
Abnormal Insulin Response in fld Cells--
We next considered the
putative functions of the Ifld1 and Ifld2
proteins, together with those encoded by other RDA sequences that we
had isolated (Table I). Both Ras-related proteins (Ifld1) and Ser/Thr kinases (Ifld2) are potentially involved in
hormone signal transduction; interestingly, insulin-like growth
factor-binding protein-4 (RDA clone W18), the G protein 
-subunit
(F26), and the F24 sequence with similarity to a guanine nucleotide
regulatory protein could also potentially function in signal
transduction. A number of RDA clones with altered expression in
fld liver are known to be associated with the actin
cytoskeleton, including actin (F27), profilin (F5), -2 actinin (W2),
and myosin light chain (W1). Because GTP-binding proteins, kinases, and
the actin network are all intimately involved in the propagation of
hormone signals (20, 21), we hypothesized that the altered expression levels of these mRNAs in fld cells might be associated
with impaired capacity for hormone-induced cytoskeleton reorganization.
To test this, we directly examined the actin cytoskeleton response to
acute treatment with serum and insulin in primary cells isolated from
wild type and fld mice. Both serum and insulin induce characteristic changes in cytoskeleton architecture in normal cells
(22, 23). The lysophosphatidic acid present in serum induces the
formation of elongated actin stress fibers, whereas insulin induces the
formation of protrusions around the periphery of cells known as
membrane ruffles. To determine whether cytoskeletal response to either
of these stimuli was impaired in fld cells, we isolated
preadipocytes from fld and wild type mice, serum starved them to produce a quiescent state, and then treated them with 1% serum
or 40 ng/ml insulin for 10 min to induce cytoskeletal rearrangement
(22, 23). The actin cytoskeleton was then visualized by staining with
fluorescently tagged phalloidin, a fungal toxin that specifically binds
actin. As shown in Fig. 4, the addition of serum to preadipocytes from both wild type and fld mice
resulted in the formation of elongated actin stress fibers visible
within the cytosol (Fig. 4, a and b). In
contrast, treatment of wild type cells with insulin elicited the
formation of membrane ruffles, which result from polymerization of a
meshwork of actin filaments at the cell surface (Fig. 4c).
Remarkably, membrane ruffles were not formed upon insulin treatment of
preadipocytes from fld mice (Fig. 4d). These
results suggest that fld cells are not generally deficient
in actin polymerization but that the propagation of an insulin signal
through the cytoskeleton is impaired and provides a functional link
with the observed alterations in mRNA levels for actin and
associated proteins. Further studies will be required to determine
whether the impaired hormone response of the cytoskeleton represents a
direct or secondary manifestation of the underlying fld
mutation.
View larger version (108K):
[in this window]
[in a new window]
|
Fig. 4.
Response of actin cytoskeleton to serum and
insulin in cells isolated from wild type and fld
mice. Preadipocytes were isolated from the inguinal fat pads
of 4-week-old wild type and fld mice and grown in culture
(see "Experimental Procedures"). Cells were brought to a quiescent
state by serum starvation and then treated for 5 min with 1% serum
(a and b) or 40 ng/ml insulin (c and
d), followed by fixation and staining with fluorescent
tagged-phalloidin. Wild type cells exhibited stress fiber formation in
response to serum (a), and membrane ruffles in response to
insulin (c). Cells from fld mice also formed
stress fibers in response to serum (b) but failed to exhibit
membrane ruffles after insulin treatment (d). Magnification:
a and b, 630×; c and d,
1000×.
|
|
| |
DISCUSSION |
The phenotype of an organism or tissue can ultimately be traced
back to the set of genes expressed in individual cells. Thus, the
characterization of differences in mRNA expression between cell
types provides a window into the fundamental differences in cellular
function that may exist between them. Using the RDA technique, we have
isolated several mRNAs with differential expression patterns in the
fatty liver of fld mutant mice compared with their wild type counterparts. RDA has previously been utilized for comparison of expression patterns in cultured cells undergoing different treatments (8-11); the current study further establishes RDA as a
reliable technique to isolate differentially expressed sequences from animal tissues as a means to compare wild type and mutant phenotypes.
The expression levels for mRNAs isolated by RDA included species of
high, moderate, and low abundance and both large and modest differences
in hepatic expression levels between fld and wild type mice
(summarized in Table I). Overall, differential mRNA expression
levels were confirmed for ~70% of the RDA clones isolated here,
indicating a success rate for RDA that compares favorably to related
techniques for differential mRNA cloning (24, 25). Most of the
remaining clones isolated corresponded to mRNAs that could not be
detected on Northern blots containing total RNA, indicating that they
may be expressed at low levels. Preliminary experiments performed with
primers designed to two of these sequences have allowed detection using
reverse transcriptase-coupled PCR (data not shown). Most striking in
terms of relative mRNA levels in fld versus
wild type were the three sequences Ifld1, Ifld2, and Ifld3, which exhibited vastly elevated expression levels
in fld in contrast to barely detectable expression in wild
type tissue.
A potential limitation of the RDA technique is that it does not reveal
whether differential expression at the mRNA level is ultimately
reflected in the corresponding protein levels. Clearly it is not
feasible to directly examine protein levels for the majority of RDA
products, many of which are novel, but we have confirmed that protein
levels for two of the RDA products do indeed increase in parallel with
the elevated mRNA levels. Using quantitative two-dimensional gel
electrophoresis, we previously demonstrated that apo A-IV and actin
protein levels are increased 7- and 2-fold, respectively, in liver from
neonatal fld compared with wild type mice (5). In these
two-dimensional electrophoresis studies, we detected nearly two dozen
additional unidentified proteins with altered expression levels in the
fld fatty liver; it is possible that some of these
correspond to additional RDA products isolated in the current studies.
Of primary significance in our analysis of differential gene expression
in fld cells is the novel insight it has provided into the
nature of the metabolic defect resulting from the fld gene
mutation. Our studies revealed altered gene expression levels for
several proteins that may be implicated in insulin signaling and
cytoskeleton dynamics. For example, Ifld1, which is
dramatically induced in the fld fatty liver, shares
approximately 60% amino acid identity to a mouse Rac protein,
including conservation of GTP-binding and effector motifs, and
Ifld2 was identified as a putative novel Ser/Thr protein
kinase based on 75% identity to members of this family (Fig. 2). Both
Rac and Ser/Thr protein kinases are involved in insulin signal
transduction and the resulting cytoskeletal rearrangement; Rac is
absolutely required for membrane ruffle formation (23), and its effect
may be mediated by Ser/Thr kinases such as p21-activated protein
kinases (26). Furthermore, a number of additional RDA clones encode
proteins associated with the actin cytoskeleton. These include several
proteins (i.e. actin, profilin, -actinin, and myosin)
that are known to be localized to membrane ruffles (27-29). Although
our genetic mapping results excluded Ifld1,
Ifld2, and these cytoskeletal components as the fld gene, the altered mRNA expression levels detected in
fld cells may represent a compensatory response for the
nonfunctional fld gene, indicating that the fld
gene product may be required for development of normal insulin
response. Consistent with this interpretation is our recent finding
that fld mice are hyperinsulinemic and glucose intolerant,
indicating impaired response of fld tissues to
insulin.2
The RDA clones characterized in this study were all isolated from
liver, but two of the novel sequences that were isolated (Ifld1 and Ifld2) were also found to be expressed
in two other tissues that are clearly affected by the fld
mutation, adipose tissue, and peripheral nerve. In both adipocytes and
peripheral nerve Schwann cells, signaling pathways triggered by insulin
and/or insulin-like growth factors are crucial for the regulation of differentiation and metabolism (30-34). Our novel finding of impaired insulin response in fld cells may therefore represent a
direct causal link to the phenotype of fld adipocytes and
Schwann cells. The signaling pathways involved in insulin-mediated
cytoskeleton dynamics and membrane ruffle formation and the gene
products involved in this biochemical cascade have not been fully
elucidated. The identification of the fld gene defect may
therefore shed light on molecular events involved in development of
normal insulin response.
| |
ACKNOWLEDGEMENTS |
We thank Yu-Rong Xia and Jake Lusis (UCLA)
for assistance in genomic mapping of RDA clones. We thank David Schatz
(Yale) for providing a detailed protocol for cDNA-RDA.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL28481.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF054279 (Ifld1), AF054280 (Ifld2),
AF054281 (Ifld3), AF054282 (F23), and AF054283 (F24).
§
Supported by Research Fellowship Kl 973/3-1 from the Deutsche
Forschungsgemeinschaft. Present address: Dept. of Biology,
Philipps-University, D-35043, Marburg, Germany.
Present address: Dept. of Medicine, Columbia University
College of Physicians and Surgeons, New York, NY 10032.
**
Recipient of an American Heart Association Established Investigator
Award. To whom correspondence should be addressed: West Los Angeles VA
Medical Center, 11301 Wilshire Blvd., Bldg. 113, Rm. 312, Los Angeles,
CA 90073. E-mail: Reuek@ucla.edu.
2
K. Reue, manuscript in preparation.
| |
ABBREVIATIONS |
The abbreviations used are:
fld, fatty liver dystrophy;
RDA, representational difference analysis;
Chr, chromosome;
PCR, polymerase chain reaction;
EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.
| |
REFERENCES |
| 1.
|
Langner, C. A.,
Birkenmeier, E. H.,
Ben-Zeev, O.,
Schotz, M. C.,
Sweet, H. O.,
Davisson, M. T.,
and Gordon, J. I.
(1989)
J. Biol. Chem.
264,
7994-8003[Abstract/Free Full Text]
|
| 2.
|
Langner, C. A.,
Birkenmeier, E. H.,
Roth, K. A.,
Bronson, R. T.,
and Gordon, J. I.
(1991)
J. Biol. Chem.
266,
11955-11964[Abstract/Free Full Text]
|
| 3.
|
Rowe, L. B.,
Sweet, H. O.,
Gordon, J. I.,
and Birkenmeier, E. H.
(1996)
Mamm. Genome
7,
555-557[CrossRef][Medline]
[Order article via Infotrieve]
|
| 4.
|
Reue, K.,
and Doolittle, M. H.
(1996)
J. Lipid Res.
37,
1387-1405[Abstract]
|
| 5.
|
Rehnmark, S.,
Giometti, C. S.,
Slavin, B. G.,
Doolittle, M. H.,
and Reue, K.
(1998)
J. Lipid Res.
39,
2209-2217[Abstract/Free Full Text]
|
| 6.
|
Lisitsyn, N. A.,
Lisitsyn, N. M.,
and Wigler, M. H.
(1993)
Science
259,
946-951[Abstract]
|
| 7.
|
Lisitsyn, N. A.,
Lisitsina, N. M.,
Dalbagni, G.,
Barker, P.,
Sanchez, C. A.,
Gnarra, J.,
Linehan, W. M.,
Reid, B. J.,
and Wigler, M. H.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
151-155[Abstract/Free Full Text]
|
| 8.
|
Braun, B. S.,
Frieden, R.,
Lessnick, S. L.,
May, W. A.,
and Denny, C. T.
(1995)
Mol. Cell Biol.
15,
4623-4630[Abstract]
|
| 9.
|
Chu, C. C.,
and Paul, W. E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2507-2512[Abstract/Free Full Text]
|
| 10.
|
Edman, C. F.,
Prigent, S. A.,
Schipper, A.,
and Feramisco, J. R.
(1997)
Biochem. J.
323,
113-118
|
| 11.
|
Hubank, M.,
and Schatz, D. G.
(1994)
Nucleic Acids Res.
22,
5640-5648[Abstract/Free Full Text]
|
| 12.
|
Labarca, C.,
and Paigen, K.
(1980)
Anal. Biochem.
102,
344-352[CrossRef][Medline]
[Order article via Infotrieve]
|
| 13.
|
Cohen, R. D.,
Castellani, L. W.,
Qiao, J.-H.,
Van Lenten, B. J.,
Lusis, A. J.,
and Reue, K.
(1997)
J. Clin. Invest.
99,
1906-1916[Medline]
[Order article via Infotrieve]
|
| 14.
|
Altschul, S. F.,
Gish, W.,
Miller, W.,
Myers, E. W.,
and Lipman, D. J.
(1990)
J. Mol. Biol.
215,
403-410[CrossRef][Medline]
[Order article via Infotrieve]
|
| 15.
|
Don, R. H.,
Cox, P. T.,
Wainwright, B. J.,
Baker, K.,
and Mattick, J. S.
(1991)
Nucleic Acids Res.
19,
4008[Free Full Text]
|
| 16.
|
Welch, C. L.,
Xia, Y.-R.,
Schecter, I.,
Farese, R.,
Mehrabian, M.,
Mehdizadeh, S.,
Warden, C. H.,
and Lusis, A. J.
(1996)
J. Lipid Res.
37,
1406-1421[Abstract]
|
| 17.
|
Manly, K. F.
(1993)
Mamm. Genome
4,
303-313[CrossRef][Medline]
[Order article via Infotrieve]
|
| 18.
|
Briquet-Laugier, V.,
Dugail, I.,
Ardouin, B.,
Le Liepvre, X.,
Lavau, M.,
and Quignard-Boulange, A.
(1994)
Am. J. Physiol.
267,
E439-E446[Abstract/Free Full Text]
|
| 19.
|
Ridley, A. J.
(1995)
Methods Enzymol.
256,
306-313[Medline]
[Order article via Infotrieve]
|
| 20.
|
Gupta, P. D.,
Nandini, R.,
and Rao, K. S.
(1996)
Cytobios
86,
75-111[Medline]
[Order article via Infotrieve]
|
| 21.
|
Tsakiridis, T.,
Wang, Q.,
Taha, C.,
Grinstein, S.,
Downey, G.,
and Klip, A.
(1997)
Soc. Gen. Physiologists Series
52,
257-271
|
| 22.
|
Ridley, A. J.,
and Hall, A.
(1992)
Cell
70,
389-399[CrossRef][Medline]
[Order article via Infotrieve]
|
| 23.
|
Ridley, A. J.,
Paterson, H. F.,
Johnston, C. L.,
Diekmann, D.,
and Hall, A.
(1992)
Cell
70,
401-410[CrossRef][Medline]
[Order article via Infotrieve]
|
| 24.
|
Sunday, M. E.
(1995)
Am. J. Physiol.
269,
L273-L284[Abstract/Free Full Text]
|
| 25.
|
Wan, J. S.,
Sharp, S. J.,
Poirier, G. M.-C.,
Wagaman, P. C.,
Chambers, J.,
Pyati, J.,
Hom, Y.-L.,
Galindo, J. E.,
Huvar, A.,
Peterson, P. A.,
Jackson, M. R.,
and Erlander, M. G.
(1996)
Nature Biotechnol.
14,
1685-1691[CrossRef][Medline]
[Order article via Infotrieve]
|
| 26.
|
Dharmawardhane, S.,
Sanders, L. C.,
Martin, S. S.,
Daniels, R. H.,
and Bokoch, G. M.
(1997)
J. Cell Biol.
138,
1265-1278[Abstract/Free Full Text]
|
| 27.
|
Machesky, L. M.,
and Pollard, T. D.
(1993)
Trends Cell Biol.
3,
381-385
[CrossRef][Medline]
[Order article via Infotrieve] |
| 28.
|
Bretscher, A.,
and Lynch, W.
(1985)
J. Cell Biol.
100,
1656-1663[Abstract/Free Full Text]
|
| 29.
|
Conrad, P. A.,
Giuliano, K. A.,
Fisher, G.,
Collins, K.,
Matsudaira, P. T.,
and Taylor, L. D.
(1993)
J. Cell Biol.
120,
1381-1391[Abstract/Free Full Text]
|
| 30.
|
Spritz, N.,
Singh, H.,
and Marinan, B.
(1975)
J. Clin. Invest.
55,
1049-1056
|
| 31.
|
Cheng, H. L.,
and Feldman, E. L.
(1997)
J. Cell. Physiol.
171,
161-167[CrossRef][Medline]
[Order article via Infotrieve]
|
| 32.
|
Ong, J. M.,
Kirchgessner, T. G.,
Schotz, M. C.,
and Kern, P. A.
(1988)
J. Biol. Chem.
263,
12933-12938[Abstract/Free Full Text]
|
| 33.
|
Semenkovich, C. F.,
Wims, M.,
Noe, L.,
Etienne, J.,
and Chan, L.
(1989)
J. Biol. Chem.
264,
9030-9038[Abstract/Free Full Text]
|
| 34.
|
Raynolds, M. V.,
Awald, P. D.,
Gordon, D. F.,
Gutierrez-Hartmann, A.,
Rule, D. C.,
Wood, W. M.,
and Eckel, R. H.
(1990)
Mol. Endocrinol.
4,
1416-1422[CrossRef][Medline]
[Order article via Infotrieve]
|
| 35.
|
Saraste, M.,
Sibbald, P. R.,
and Wittinghofer, A.
(1990)
Trends Biochem. Sci.
15,
430-434[CrossRef][Medline]
[Order article via Infotrieve]
|
| 36.
|
Calés, C.,
Hancock, J. F.,
Marshall, C. J.,
and Hall, A.
(1988)
Nature
332,
548-551[CrossRef][Medline]
[Order article via Infotrieve]
|
| 37.
|
Pinna, L. A.
(1990)
Biochim. Biophys. Acta
1054,
267-284[Medline]
[Order article via Infotrieve]
|
| 38.
|
Woodgett, J. R.,
Gould, K. L.,
and Hunter, T.
(1986)
Eur. J. Biochem.
161,
177-184[Medline]
[Order article via Infotrieve]
|
| 39.
|
Kishimoto, A.,
Nishiyama, K.,
Nakanishi, H.,
Uratsuji, Y.,
Nomura, H.,
Takeyama, Y.,
and Nishizuka, Y.
(1985)
J. Biol. Chem.
260,
12492-12499[Abstract/Free Full Text]
|
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
T. Kanzleiter, T. Schneider, I. Walter, F. Bolze, C. Eickhorst, G. Heldmaier, S. Klaus, and M. Klingenspor
Evidence for Nr4a1 as a cold-induced effector of brown fat thermogenesis
Physiol Genomics,
December 14, 2005;
24(1):
37 - 44.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Reue, P. Xu, X.-P. Wang, and B. G. Slavin
Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene
J. Lipid Res.,
July 1, 2000;
41(7):
1067 - 1076.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
F. M. Gregoire, Q. Zhang, S. J. Smith, C. Tong, D. Ross, H. Lopez, and D. B. West
Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice
Am J Physiol Endocrinol Metab,
March 1, 2002;
282(3):
E703 - E713.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. BOEUF, M. KLINGENSPOR, N. L. W. VAN HAL, T. SCHNEIDER, J. KEIJER, and S. KLAUS
Differential gene expression in white and brown preadipocytes
Physiol Genomics,
October 10, 2001;
7(1):
15 - 25.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
