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Department of Endocrinology and Metabolism C, Aarhus University Hospital (J.X., S.G., K.H.), 8000 Aarhus C, Denmark; and Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital (M.K., T.F.O.), 8200 Aarhus N, Denmark
Address all correspondence and requests for reprints to: Kjeld Hermansen, M.D., Department of Endocrinology and Metabolism C, Aarhus University Hospital, Tage Hansens Gade 2, 8000 Aarhus C, Denmark. E-mail: kjeld.hermansen{at}dadlnet.dk
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Introduction |
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Generally, the immediate biological response of cells to changes in the external milieu is regulated (within seconds or minutes) by the changes in enzyme activity. In contrast, the adaptation to more prolonged changes in the environment depends on the regulation of gene transcription and protein translation. Similarly, the abnormal insulin secretion secondary to chronic exposure of fatty acids may include the regulation of gene expression, protein translation, processing, and modification. Studies of fatty acid-induced changes in gene expression in islets or cultured ß-cells have focused only on a few candidate genes that are thought to be important for the adaptation (5, 6, 7, 8). It is, however, likely that chronic exposure to fatty acid induces changes in pancreatic ß-cell function resulting from changes not only in one or a few genes, but in a multitude of genes.
To gain further insight into the mechanism of changes in ß-cell function after chronic exposure to fatty acids we have looked at the chronic effects of palmitate on the clonal insulin-producing cell line, INS-1, which was cultured for up to 44 d. Unlike the previous study we used 50 and 200 µM palmitate (which are relatively low concentrations) and 0.5% BSA in our studies to prevent a direct toxic effect by unbound fatty acids. The pattern of gene expression changes was studied using gene chip analysis (GCA), which directly monitors the expression levels of 8740 genes in parallel (9, 10).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
The passage number of INS-1 cells (provided by Prof. C. B.
Wolheim, Geneva, Switzerland) used in this study was 61–67. The
approximate time for doubling the cell number was 2–3 d. Before the
study INS-1 cells were maintained at 10 mM glucose in a
modified RPMI 1640 medium (see below) and passed weekly
(11). The INS-1 cells were grown in a humidified
atmosphere (5% CO2, 95% air at 37 C) in
monolayer in a modified RPMI 1640 medium in flasks or 24-well plates
(Nunc Brand Products, Nunc A/S, Roskilde, Denmark). We supplemented the
medium with 10% (vol/vol) FCS, 6.6 mM glucose, 10
mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1
mM sodium pyruvate, 2 mM
L-glutamate, 50 µM ß-mercaptoethanol (all
from Life Technologies, Inc., Paisley, UK) ,and 0.5% BSA
(fatty acid free; Roche Molecular Biochemicals, Mannheim,
Germany). A stock solution of palmitate (Sigma, St. Louis,
MO) was prepared by mixing and heating to 90 C with equal molar amounts
of NaOH and palmitic acid and was supplemented with distilled water to
a concentration of 400 mM. It was diluted further with 5%
BSA (fatty acid free) to a concentration of 50 mM,
sterilized with a filter, and stored at 4 C. A suitable amount warm
palmitate (
40 C) was slowly added to 37 C culture medium to make
final concentrations of 50 µM (P50) and 200
µM (P200) palmitate. As a control, cells were cultured
without palmitate. As a standard procedure, culture medium was changed
three times every week. The cells were passaged weekly into new flasks
and cultured continuously under similar conditions. Simultaneously,
cells were subcultured for 48 h in 24-well plates for secretion
studies.
Insulin secretion study
Cells were precultured in a modified Krebs-Ringer buffer (125
mM NaCl, 5.9 mM KCl, 1.28 mM
CaCl2, 5.0 mM
NaHCO3, and 25 mM HEPES, pH 7.4; all
from Sigma) containing 3.3 mM glucose and
0.1% BSA at 37 C for 30 min. After preculture, glucose-stimulated
insulin secretion was determined by incubating the cells in 1.0 and
16.7 mM glucose for 60 min. For fatty acid stimulation, 400
µM palmitate was added in presence of 16.7 mM
glucose for 60 min. After incubation, 300 µl medium were collected
and centrifuged, and 200 µl supernatant were stored at -20 C for
insulin assays.
Protein assay
The remaining medium was discarded after collecting samples for
insulin assay. Cells were washed with cold PBS and lysed in 0.1
M NaOH; the total protein was measured by a
detergent-compatible protein kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Insulin content measurement
Before every cell passage, the cells were harvested after
trypsin treatment (Life Technologies, Inc.), centrifuged,
and counted. Cells (2 x 105) were
separately collected for protein and insulin measurements. The cells
were transferred to glycine-BSA buffer (100 mM glycine and
0.25% BSA, pH 8.8), and insulin was released by sonication (Sonifier
250, Branson, Danbury, CT) on ice twice for 15 sec each time. After
centrifugation for 30 min at 16,000 rpm, the supernatant was collected
and frozen at -20 C for later assay, and the protein concentration in
the same collection of cells was determined as described above. The
insulin content was adjusted to the protein concentration.
Insulin assay
Insulin was measured by RIA with a guinea pig, anti-porcine
insulin antibody, mono-125I- (Tyr-A14)-labeled
human insulin as tracer, and rat insulin as standard. Free and bound
radioactivity were separated using ethanol. The inter- and intraassay
variation coefficients were both less than 5%.
RNA extraction
Total RNA was isolated from the INS-1 cells using TRIzol
(Life Technologies, Inc.).
Gene expression chip analysis
In present study the expression of 8740 genes or expressed
sequence tags (ESTs) was investigated by the following steps.
mRNA preparation. To ensure that the samples were representative and to reduce preanalyzed variation, three extracted total RNA samples were pooled from the same time point and culture conditions in an equal amount. One hundred micrograms of total RNA were used for mRNA preparation. Polyadenylated RNA was isolated by an oligo(deoxythymidine) selection step (Oligotex mRNA kit, QIAGEN, Valencia, CA).
cRNA preparation. One microgram of mRNA was used as starting material for the cDNA preparation. The first and second strand cDNA synthesis was performed using the SuperScript Choice System (Life Technologies, Inc., Roskilde, Denmark) basically according to the manufacturer’s instructions, but using an oligo(deoxythymidine) primer containing a T7 RNA polymerase promoter site. Labeled cRNA was prepared using the MEGAscript in vitro transcription kit (Ambion, Inc., Austin, TX). Biotin-labeled CTP and UTP were used together with unlabeled NTPs in the reaction. After the in vitro transcription reaction, the unincorporated nucleotides were removed using RNeasy columns (QIAGEN).
Array hybridization and scanning
Ten micrograms of cRNA were fragmented at 94 C for 35 min in a
fragmentation buffer containing 40 mM Tris-acetate (pH
8.1), 100 mM KOAc, and 30 mM MgOAc. Before
hybridization, the fragmented cRNA was heated to 95 C for 5 min in a
6 x SSPE-T hybridization buffer [1 M NaCl, 10
mM Tris (pH 7.6), and 0.005% Triton] and subsequently to
40 C for 5 min before loading onto the Affymetrix probe array cartridge
rat U34A gene chip. The probe array was then incubated for 16 h at
40 C at constant rotation (60 rpm).
The washing and staining procedure was performed in the Affymetrix Fluidics Station (9). The probe array was exposed to 10 washes in 6 x SSPE-T at 25 C, followed by 4 washes in 0.5 x SSPE-T at 50 C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate (10 mg/ml; Molecular Probes, Inc., Eugene, OR) in 6 x SSPE-T for 30 min at 25 C, followed by 10 washes in 6 x SSPE-T at 25 C. The probe arrays were scanned at 560 nm by a scanning confocal microscope with an argon ion laser as the excitation source (made for Affymetrix by Hewlett-Packard Co., Palo Alto, CA).
Data analysis
The readings from the quantitative scanning were analyzed by
Affymetrix gene chip analysis software (9). More than
300,000 probes were used in the chip. For each gene or EST,
approximately 20 pairs of probes (complemented to different parts of
mRNA of a gene) were used to measure the level of expression. One pair
of probes consists of a perfect match (PM) probe and a mismatch (MM)
probe (one MM nucleotide in the middle of sequence). The later is used
to determine the background signal after hybridization. A probe pair is
considered positive when the intensity of PM minus the intensity of MM
and the ratio of the intensity of PM to the intensity of MM are greater
than or equal to the statistical threshold. By calculating the
intensity of all pairs of probes, the level of gene expression was
determined. By comparing the difference in intensity of all pairs of
probes between samples for each gene, the difference of expression was
defined. Detailed protocols for data analysis and extensive
documentation of the sensitivity and quantitative aspects of the method
have been described previously (9).
Confirmation of gene expression with real-time RT-PCR
To confirm our findings by GCA, we investigated the expressions
of carnitine palmitoyl transferase-1 (CPT-1), insulin receptor (IR),
insulin receptor substrate-2 (IRS-2), glucokinase (GK), type 1
hexokinase (HK1), and insulin 1 gene by real-time RT-PCR. cDNA was made
using random hexamer primers as described by the manufacturer (GeneAmp
RNA PCR kit, Perkin-Elmer Corp./Cetus, Norwalk, CT). Then,
a PCR master mixture containing the specific primers (Table 1
) and AmpliTaq Gold DNA
polymerase were added. Real-time quantitation of the target genes to
ß-actin mRNA was performed with an SYBR-Green real-time PCR assay
using an ICycler PCR machine from Bio-Rad Laboratories, Inc. (12, 13). Briefly, each gene was amplified in
separate tubes, and the increase in fluorescence was measured in
real-time. The threshold cycle (Ct), which is defined as the fractional
cycle number at which the fluorescence reaches 10x the SD
of the baseline that was calculated. The gene expression relative to
the ß- actin expression was calculated essentially as described in
User Bulletin 2, 1997, from Perkin-Elmer Corp. covering
the aspect of relative quantitation of gene expression. In short, the
target gene to ß-actin mRNA expression was calculated for each sample
as 1/2
C [C = (Ct target gene -
Ct ß-actin)]. All samples were amplified in triplicate. A similar
set-up was used for negative controls, except that reverse
transcriptase was omitted, and no PCR products were detected under
these conditions.
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Results |
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Basal insulin secretion and stimulated insulin secretion
BIS was defined as insulin secretion during 60-min incubation in
the presence of 1.0 mM glucose. Although BIS did not change
in cells cultured in palmitate for 2 d, it increased after d 9 and
reached a maximum on d 44 in cells cultured with P200 (compared with
other time points, data not shown). On d 44, BIS was increased 3-fold
after culture with P50 and 4.5-fold with P200, respectively
(P < 0.05; Fig. 1A).
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When expressed in relative terms (fold increase compared with the
release at 1.0 mM glucose), enhanced GSIS and FSIS were
observed on d 2 in cells exposure to palmitate, whereas they displayed
the lowest values in cells cultured with P200 on d 44 (Fig. 1B).
Insulin content
The insulin content in the INS-1 cells was not significantly
changed after exposure to either P50 or P200 for 44 d (1827
± 496 and 2504 ± 435; control, 2086 ± 185 ng/mg protein;
n = 6 for each group; P > 0.05).
GCA
General pattern of gene expression changes. The number of
genes that changed expression depended on the cut-off values set
(significant level). The cut-off values are arbitrary. The larger the
numerical sort score (SS; a value based on both fold change and average
difference change), the more significant or reliable is the measured
difference in expression of gene between the experimental group and the
control group. If fold change were set at 2, and SS to 0.5, the number
of genes that changed was 507. However, changing the SS to 0.65 and the
fold change to 3, the number of affected genes was reduced to 345
(Table 2). In the present study genes or
ESTs with an SS of 0.65 or more, with the difference call being
increased or decreased, and showing 2-fold or more changes were
considered to be significant changes (14). The number of
genes showing significant changes at the expression levels is given in
Fig. 2. In general, down-regulated genes
were more common than up-regulated genes. The changes in response to
palmitate exposure were dose and time dependent.
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Confirmation by RT-PCR (Fig. 3)
The same expression pattern as that identified with GCA was found
by real-time RT-PCR for CPT-1, IR, IRS-2, GK, and insulin 1. Type 1
hexokinase in INS-1 cells was very low (absent), and no difference
between groups was found by GCA. However, it was down-regulated by
palmitate on both d 2 and 44 using RT-PCR.
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Discussion |
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A striking finding is the increased insulin secretion at low glucose (BIS). BIS was higher in cells cultured in P200 than in P50 on d 2. It increased on d 9 after culture in P200, whereas an increase first occurred from d 37 after culture in P50 (our unpublished data). This indicates that both exposure time and fatty acid concentrations are crucial for eliciting elevated BIS. Increased BIS is one of the characteristic features of lipotoxicity in addition to the decline in blunted GSIS (24). Hyperinsulinemia compensates the insulin resistance in the early stage of the insulin resistance syndrome (25, 26, 27), and it has also frequently been found in early stages of type 2 diabetes (28). Fatty acid-induced insulin resistance is compensated by increased GSIS at the beginning. However, studies in animals indicate that early hyperinsulinemia seems by itself to be able to cause insulin resistance and, finally, dysfunction of ß-cells (29). It can be argued that the glucose level of 1 mM is a very low basal glucose concentration. However, it is difficult to decide what is a normal basal glucose level in clonal ß-cells. We have compared the impact of 1 and 3.3 mM glucose in previous and recent experiments and have uniformly found similar response patterns to different stimuli, including high glucose, and similar insulin secretion at 1.0 and 3.3 mM glucose (unpublished results). No significant decrease in total insulin release occurred during stimulation after prolonged culture in the presence of P200. However, we found that the ß-cell responsiveness in relative terms (fold change) to glucose and palmitate was blunted. This apparently contrasts with other studies (29) in which islet cells that have been exposed long-term to higher fatty acid concentrations (e.g. 0.4 and 1 mM) or without BSA supplement showing suppressed GSIS. Using similar pharmacological doses of palmitate of 0.4 and 1.0 mM, we also found suppressed stimulated insulin secretion from INS-1 cells in absolute terms (unpublished results). However, we have chosen lower palmitate concentrations and 0.5% BSA in culture medium to prevent direct cytotoxicity of unbound fatty acids. Additionally, the relatively low glucose level used (6.6 mM) may also account for the lack of reduction in the absolute level of GSIS. This is supported by the findings that fatty acid inhibition of cell proliferation was only found in the presence of a stimulating glucose concentration (30). The increased BIS and declined responsiveness to nutrients (in relative, but not absolute, terms) may represent the early stage of lipotoxicity in both humans and animals. The so-called lipotoxicity in different diabetic models, for instance, the male Zucker diabetic fat rat, is characterized by increased BIS and decreased ß-cell responsiveness, i.e. less fold increase in stimulated insulin secretion compared with lean control. Concomitantly, the absolute plasma insulin level is much higher than that in lean nondiabetic controls in the early stage, although it is insufficient to compensate for the metabolic aberration (31). Furthermore, it was found that even obese female Zucker nondiabetic rats had higher glucose-stimulated insulin secretion than lean male nondiabetic rats (118 ± 19 vs.94 ± 12 µU/ml); however, the former group has a higher plasma glucose concentration (9.15 ± 0.02 vs. 5.70 ± 0.17 mM). After calculating the fold increase, it is not surprising to find that the former group had a much lower responsiveness (0.8- vs. 12-fold) (31). At a later stage of disease, the absolute insulin concentration also declines. Therefore, we propose that the increased BIS and the declined responsiveness (but not the absolute insulin secreted) to nutrient stimulation are early characteristics of lipotoxicity.
GCA is being used more and more widely as a method for providing a
broad picture of the state of the cell by monitoring the expression
level of thousands of genes (14, 32, 33). A cut-off value
determines genes that changed significantly. To confirm the findings
regarding gene expression detected with GCA, six genes were further
characterized by real-time RT-PCR. All of these genes (CPT-1, IR, and
IRS-2) that were found to be changed by GCA techniques were confirmed
by RT-PCR (Fig. 3), and no difference in the expression of GK and
insulin 1 genes was found by either method. However, a few differences
between the two techniques were observed. The IRS-2 expression on d 2
was not significantly down-regulated using the GCA, whereas the
decrease in expression was significant using the RT-PCR technique. It
is interesting that the patterns of expression of IRS-2 using the two
different techniques are similar. Finally, HK1 expression seemed to be
very different using the two techniques; however, HK1 expression was
very low, and actually using the GCA it was described as absent. Using
RT-PCR (which is much more sensitive), the expression could easily be
measured and was found to be down-regulated. Palmitate is a PPAR
agonist, inducing the expression of genes involved in fatty acid
oxidation (34). The increased expression of CPT-1,
long-chain acyl CoA dehydrogenase, and other genes involved in
fatty acid oxidation in the present study corroborated this report. The
down-regulation of the c-erb-A TR (35) and the
RAR (36), which are transcription factors that inhibit the
expression of genes involved in fatty acid oxidation, may contribute to
the up-regulation. In addition, the down-regulation of gastrin-binding
protein may lead to the enhanced fatty oxidation (37). On
the other side, palmitate may suppress the effect of PPAR
by
inhibiting the expression of adipocyte differentiation and determinator
1 and result in a suppression of fatty acid synthetase and acetyl-CoA
carboxylase, as seen in adipocytes (36). These changes in
gene expression may reflect the adaptation of cells to fatty acid
exposure, i.e. induced expression of genes regulating fatty
acid oxidation and suppressed expression of genes regulating fatty acid
synthesis (6, 8, 29, 38). It suggests that a compensatory
fatty acids oxidation takes place during palmitate exposure.
Interestingly, it seems that the changes in genes involved in fatty
acid oxidation on d 2 were more striking than the changes on d 44.
Thus, an increased expression of peroxisomal enoyl hydratase-like
protein, 2,4-dienoyl-CoA reductase was found only on d 2. The
expression of CPT-1, epoxide hydrolase, and 3-oxoacyl-CoA thiolase gene
increased 2 times more on d 2 than on d 44. The lack of a proportional
increase in fatty acid oxidation may result in triglyceride
accumulation within cells, which is supposed to relate to apoptosis
(3). We did not determine cell triglyceride content;
therefore, whether this difference contributed to the functional
differences between d 2 and d 44 remains to be elucidated. Palmitate
was shown to suppress the expression of stearoyl-CoA desaturase-2
(SCD-2) and SCD-2 homologue gene in INS-1 cells. SCD-2 can transform
the saturated fatty acid into unsaturated fatty acid and thereby change
plasma membrane fluidity (39). The physiological
importance of our observation is as yet unclear.
The expression of HK1 was absent by GCA, and it was found to be very
low using RT-PCR, i.e. the mRNA (ratios to ß-actin) were
4 x 10-6–10-3 and
7 x 10-3–9 x
10-3 for HK1 and GK, respectively (data not
shown). The expression of the HK1 gene was suppressed during palmitate
exposure, suggesting that HK1 may not play a major role in the enhanced
BIS in the current study. However, caution should be taken because
clonal INS-1 cells may not respond to fatty acids in the same way as
normal ß-cells or islets (2, 3). We have not found any
differences in glucokinase and glucose transporter-2 mRNA levels
between groups. In the cascade of glycolysis, aldolase A, PFK, and
ATP-citrate lyase were suppressed by long-term exposure and/or the
higher palmitate level. The up-regulation of fructose-1,6-bisphophatase
may also inhibit glycolysis, because it counteracts the reaction
catalyzed by PFK. The down-regulation of PFK is consistent with results
from studies of the heart exposed to fatty acids (40).
Except for cells cultured at P50 for 2 d, the other groups shared
similar alteration of expression of genes involved in glycolysis. This
suggested that this modification was not an exclusive mechanism
underlying the blunted insulin secretion. We found that HNF-4 gene
expression was inhibited after palmitate exposure in a time- and
dose-dependent manner. A mutation in the HNF-4 gene is responsible
for the autosomal dominant, early-onset form of type 2 diabetes
mellitus, the so-called MODY1. It was showed that the MODY1
mutant protein has lost its transcriptional trans-activation
activity (41), which affects several genes whose
expression is dependent upon HNF-4, including glucose transporter-2,
the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate
dehydrogenase, and the liver pyruvate kinase (41). We
found that aldolase A, but not aldolase B, was suppressed accompanying
the down-regulation of HNF-4. The down-regulation of genes involved in
the glycolysis and up-regulation of fatty acid oxidation in cells
cultured in fatty acid suggests that the fatty acid-glucose reciprocal
cycle (Randle cycle) (42) is operative on gene expression
levels in INS-1 cells.
Glycogen accumulation in muscle may relate to insulin resistance. It was found that troglitazone decreased the skeletal muscle glycogen content to normal and increased glycogen turnover in the Zucker diabetic fatty rat accompanying improved insulin sensitivity (43). The insulin-sensitizing action of exercise may be ascribed to enhanced glucose transporter-4 translocation and the depletion of glycogen in muscle (44). The down-regulation of glycogen phosphorylase observed in this study after culture for 44 d may result in glycogen accumulation and contribute to the insulin resistance and abnormal insulin secretion in INS-1 cells.
Our data suggest that changes in insulin mRNA transcription and insulin biosynthesis may not represent the major defects in abnormal insulin secretion after chronic palmitate exposure. However, we do not know whether a lack of increase in insulin gene expression and insulin content in parallel with the enhanced BIS also reflects a relatively insufficient insulin gene transcription and synthesis in cells cultured in P200 (7, 45). A suppression of IR gene expression after 44-d culture at P200 is accompanied by the suppression of IRS-2 and cytokine-inducible Src homology-2-containing protein. The IR and its principal substrates, IRS-1 and IRS-2, play key roles in insulin signal transduction. Recent studies suggest that secreted insulin acts directly on ß-cells, via its own receptor, to enhance insulin production in an autocrine feedforward loop (46). After specific knockout of the IR gene in the ß-cells, these mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance (47). Interestingly, IRS-2 knockout mice show severe insulin resistance with simultaneous reduction of islet size and insulin content. Consequently, ß-cells lacking IRS-2 are unable to compensate for the insulin resistance, and diabetes ensues (48, 49). Thus, the noticeable down-regulation of IRS-2 by palmitate in the present study may have significant effects on cell survival. This is supported by the finding that fatty acids inhibit IGF-I-induced INS-1 cell proliferation, whereas IRS-2 plays an important role in IGF-1 signal transduction (30). Due to the importance of the insulin signaling pathway, the combined down-regulation of IR and IRS-2 may account for the suppressed response to nutrient stimulation in P200 on d 44 in this study.
Apparently, the changes in cell function are not determined only by one or a few genes, but seem to be ascribed to a series of genes. We found that the changed genes ranged from ion channels; enzymes involved in glucose, fatty acid, and amino acid metabolism; to proteins involved in signal transduction, cell growth, replication, and apoptosis (data not shown). The accumulating or synergistic effects of different genes may be important for the alteration of cell function.
The present study has been carried out in the insulinoma cell line INS-1 and not in normal ß-cells. Consequently, when interpreting the results caution should be exercised, as the possibility exists that long-term culture of INS-1 cells in high palmitate might, for instance, lead to selection variants that do not necessarily reflect normal ß-cell biology (50). Furthermore, the effect of fatty acids on ß-cell function depends on the chain length, degree of saturation, and spatial configuration, which indicates a differential influence of specific fatty acids on gene expression (4, 18, 50).
In summary, a relatively low concentration of fatty acid induced a striking increase in basal insulin secretion and a blunted insulin responsiveness in relative terms after 44 d. This may represent an early change in lipotoxicity. The abnormal secretion was associated with changes in expression in a multitude of genes. The Randle fatty acid-glucose cycle is operative at the level of gene expression. The induced expression of genes involved in fatty acid oxidation and the down-regulated gene expressions involved in insulin signal transduction and glycolysis, especially HNF-4, glycogen phosphorylase, IR, IRS-2, and genes involved in other functions, may together contribute to changes in insulin secretion of INS-1 cells exposed to palmitate.
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Acknowledgments |
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Footnotes |
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Abbreviations: BIS, Basal insulin secretion; CoA, coenzyme A; CPT-1, carnitine palmitoyl transferase-1; Ct, threshold cycle; EST, expressed sequence tag; GCA, gene chip analysis; GK, glucokinase; GSIS, as insulin secretion during 60-min incubation in response to 16.7 mM glucose; HK1, type 1 hexokinase; HNF-4, hepatic nuclear factor-4; IR, insulin receptor; IRS-2, insulin receptor substrate-2; MM, mismatch; P50, 50 µM palmitate; P200, 200, 200 µM palmitate; PFK, phosphofructokinase; PM, perfect match; SCD-2, stearoyl-CoA desaturase 2; 6 x SSPE-T hybridization buffer, 1 M NaCl, 10 mM Tris (pH 7.6), and 0.005% Triton; SS, sort score.
Received February 12, 2001.
Accepted for publication July 23, 2001.
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