(Received for publication, July 24, 1995; and in revised form, September 15, 1995)
From the
The obese gene product, leptin, regulates adiposity.
Mice homozygous for a nonfunctional obese gene become
massively obese and develop diabetes mellitus due to overeating and
increased metabolic efficiency. The cDNA sequence of obese was
recently reported (Zhang, Y., Proenca, R., Maffei, M., Barone, M.,
Leopold, L., and Friedman, J. L.(1994) Nature 372,
425-432; Correction: (1995 Nature 374, 479). We have
determined the genomic organization of the 5` end of the mouse obese gene. The coding sequence is in exons 2 and 3. A single
TATA-containing promoter was found upstream of exon 1. A minority
(probably 5%) of the obese mRNA contained an extra,
untranslated exon between exons 1 and 2. Transcription of the obese gene was detected only in adipose cells. A 762-base pair obese gene promoter driving a luciferase gene yielded abundant activity
in transiently transfected rat adipose cells in primary culture. The obese promoter was inactive in erythroid K562 cells. Deletion
of bases from -762 downstream to -161 did not affect
promoter activity in transfected adipose cells. The -161 minimal
promoter contained consensus Sp1 and CCAAT/enhancer-binding protein
(C/EBP) motifs. Cotransfection with C/EBP
(a transcription factor
important in adipose cell differentiation) caused 23-fold activation.
These data suggest that the obese promoter is a natural target
of C/EBP
.
Obesity is common in Western society, and the underlying molecular mechanisms are not well understood. Body weight is almost certainly regulated by a feedback control mechanism (see (1) ). Classic experiments with parabiotic rats showed that overeating by one animal caused its mate to starve(2) . When animals subjected to forced over- or under-feeding were returned to ad libitum feeding, they adjusted their food intake appropriately to reach the same weight as control animals(3) . If some adipose tissue is removed from a growing, chow-fed rat, the remaining adipose tissue enlarges, so that the rat attains the same total amount of body fat as an unoperated control(4) . A recent study in humans demonstrated that upon weight gain or loss, the body's energy expenditure increases or decreases, respectively, suggesting an attempt to return to the original state(5) . Taken together, these data suggest that individuals have a set point for body weight/adiposity and that a feedback control mechanism maintains the target weight.
In humans, obesity has been shown to have a large genetic component (6) . However, little is known about the specific genes that are responsible. Insight into the regulation of obesity has come from the study of the ob/ob mouse(7) . These mice grow massively obese and develop diabetes mellitus due to overeating and increased metabolic efficiency. Parabiotic animal experiments suggest that ob/ob animals are unable to make a satiety factor, but can respond to such a factor from a parabiotic mate. Similar experiments suggest that db/db mice make the factor missing in ob/ob mice, but cannot respond to it. Recently, Friedman's laboratory used positional cloning to isolate the obese gene encoding the leptin protein(8) . The obese RNA is expressed selectively in adipose tissue. Mature, secreted leptin is 146 amino acids long and is not similar in sequence to any known protein. Treatment of ob/ob mice with leptin caused reversal of the obese phenotype(9, 10, 11) . In addition, leptin treatment caused slight weight loss in wild type mice(9, 10, 11) .
Little is known about
the regulation of the obese gene. The ob/ob
mice have a
R105term nonsense mutation and a 20-fold increased obese RNA
level(8) . This suggests that these mice have an intact
mechanism to sense adiposity and to transcribe the obese gene,
but do not make functional leptin. Thus, obese is probably
subject to regulation at the level of transcription and/or RNA
stability, and comprehension of the regulation of obese will
increase our knowledge of the adiposity sensor. As a step toward
understanding the regulation of the obese gene, we have cloned
and sequenced the wild-type obese promoter and characterized
its activity in transient expression assays in primary cultures of rat
adipose cells.
A mouse epididymal fat pad cDNA library (Clontech, ML3005b) was
screened to obtain the 5` end of the obese mRNA. PCR was first
carried out (0.5-1 µl of library in 50 µl, using 1.5
mM MgCl, 0.4 µM primers, 94 °C, 4
min followed by 25 cycles of 94 °C, 2 min, 61 °C, 1 min, 72
°C, 1 min) with primers for the obese coding region (x251)
and the left (x225) or right (x226)
arms. Nested PCR on 0.5
µl used primers x249 (obese coding) and x200 (left
)
or x201 (right
), 30 cycles, and 55 °C annealing, otherwise as
above. The PCR products were cloned (PCRscript, Stratagene), and the
clones containing the longest inserts were sequenced using an Applied
Biosystems, Inc. model 373 DNA sequencer with the fluorescent primer or
dideoxy kits (Applied Biosystems). Sequence searches used BLASTN on the
NCBI server (12) and FASTA(13) .
pob-luc-RSV (p1518) was made by insertion into SpeI-cut p(-762)ob-luc of the 479-bp HgiAI/EcoRI fragment of the RSV long terminal repeat, which contains enhancer but no promoter activity (the fragment used contained multilinker-derived restriction sites at either end and was inserted as a XbaI fragment). A clone with the EcoRI end of the enhancer away from the luciferase gene was chosen.
Transient expression in K562 cells was performed as described
elsewhere. ()Briefly, 20 µg of plasmid DNA (5 µg of
luciferase plasmid, 3 µg of RSV-
-galactosidase, and 12 µg
of pUC18 carrier) were electroporated (in 400 µl of
Dulbecco's phosphate-buffered saline, 450 V, 500 microfarads,
resulting
= 7.7 ms) into 5
10
cells,
and cultured for 46-48 h before assay.
-Galactosidase assays
were performed as described(14) .
Figure 1: Map of the mouse obese gene. At the top is a restriction map of the mouse obese gene region, showing the multiple sites for BamHI, HindIII, KpnI, and XhoI and the single sites in this region for BssHII, NotI, and SalI. Below are diagrammed the intron (thin line)/exon (thick line) structure and the positions of oligonucleotide primers used in this study. The promoter is indicated by a heavy arrow, and coding regions of the exons are shown in black. The position of an alternate splice of 3 bp at the 5` end of exon 3 is indicated by an asterisk. A B1-type repetitive element in intron 2 and a dispersed repeat in the 3`-untranslated region are shaded.
To further characterize the obese mRNAs, primer extension
using an exon 2 primer was performed (Fig. 2A). In RNA
from ob/ob adipose tissue, a single strong band was observed
at 201 nucleotides, corresponding to a first exon size of
26
bp. A weak band (
20-fold less signal, but clearly visible on the
original) was observed at
290 nucleotides, the size expected for
mRNAs containing the alternate exon. With RNA from control FVB/N
mice(18) , the 201-nucleotide band was also present in adipose
tissue (at a much lower level,
20-fold weaker than in ob/ob mice). It is likely that a faint band at
147 nucleotides
results from incomplete extension by the reverse transcriptase,
although we cannot formally rule out another minor RNA specie.
Figure 2: A, primer extension to map obese transcription start sites. Primer extension was performed on 10 µg of RNA using avian myeloblastosis virus reverse transcriptase and primer x249, and then subjected to denaturing gel electrophoresis. RNA was from peritoneal fat, brain, testes, liver, kidney, or adrenal of FVB/N mice, from peritoneal fat of ob/ob mice, or yeast tRNA, as indicated. The size, in nucleotides, of MspI-digested pBR322 marker is at the left. B, RT-PCR showing tissue specificity and alternate exon use. RT-PCR was performed using RNAs from FVB/N mice and the products were electrophoresed under denaturing conditions (see ``Materials and Methods''). The left panel shows the results of 30 amplification cycles using the exon 1/alternate exon primers, the center panel contains the products of 24 amplification cycles using the exon 1/exon 2 primers, and the right panel shows the results from 30 cycles using the alternate exon/exon 2 primer set. Below each set of lanes is a diagram showing the primers and expected amplification product sizes from mRNAs without or with the alternate exon. Exon 1 is solid, exon 2 is hatched, and the alternate exon is open. Controls include no RNA or tRNA in the reverse transcription reaction and RNA from adipose tissue without reverse transcriptase (Fat, no RT). Fat-1 and Fat-2 are independently isolated RNA samples. PCR products of the predicted sizes were detected only in RNA isolated from fat. Product specificity was confirmed by restriction enzyme digestion as detailed under ``Materials and Methods.''
We used a more sensitive RT-PCR assay for further characterization of the splicing products. RT-PCR with an exon 1/exon 2 primer set yielded the 195-bp product expected for direct splicing of exons 1 and 2 (Fig. 2B, center panel). Under the conditions used, the 288-bp product expected from RNAs containing the alternate exon was not observed. However, PCR reactions specific for splicing of exon 1 to the alternate exon and of the alternate exon to exon 2 detected the expected 111- and 270-bp products, respectively (Fig. 2B, left and right panels). Both types of obese mRNA were detected only in adipose tissue, and not in the brain, testes, liver, kidney, or adrenal samples. While the PCR assay was not strictly quantitative, the alternate exon products appeared less abundant, requiring six extra cycles to be amplified to a level comparable to the product without this exon. Thus, the alternative exon is also limited to adipose tissue and is present in a minor fraction of the obese mRNAs.
The alternate exon is the second example of alternate splicing in the obese gene, the first being the variable inclusion of the first codon in exon 3(8) . Since the alternate exon sequence (Fig. 3) does not contain an ATG, inclusion of this exon does not change the predicted protein product which is initiated downstream in exon 2.
Figure 3: Sequence of the mouse obese gene. Genomic sequence surrounding the first exon is at the top, numbered relative to the start of transcription. Putative Sp1, C/EBP, and TATA sequences are underlined and so labeled. Below are the alternate exon and flanking sequence and exons 2 and 3. Exonic bases are uppercase, coding bases are italicized, and intronic bases are lowercase. The position and orientation of primers are indicated above the sequence. Restriction sites used in constructing the reporter plasmids are double underlined. GenBank accession numbers for exon 1 and the alternate exon are U36238 and U36239. The exon 2 and 3 sequences are from (8) (GenBank accession number U18812). Our intron 2 sequence agrees with GenBank accession number U22421.
The obese gene mRNA has
been detected only in adipose tissues. We examined the tissue
specificity of the obese promoter using transient expression
in the erythroid K562 cell line (22) . To allow comparison of
the adipose cell and K562 data, the results were expressed relative to
pCIS-luciferase, a highly expressed plasmid that is active in both
types of cells (Table 1). In K562 cells, the promoterless
reporter plasmids expressed luciferase at 0.01% of the level of
pCIS-luciferase. Addition of the obese promoter did not
increase luciferase above this background. The level of expression of
p(-762)ob-luc, after normalization to pCIS-luciferase, was
162-fold greater in adipose cells than in K562 cells. Thus, there is
sufficient information contained in the 762-bp promoter to allow
expression in adipose cells, but not in K562 cells.
To map its functional regions, various lengths of the obese promoter were tested for their ability to drive the expression of a luciferase reporter gene (Fig. 4A). Deletion of regions upstream of -161 did not reduce the promoter activity. We conclude that the region up to -161 functions as a promoter in adipose cells and that addition of another 600 bp of upstream DNA did not increase the promoter activity.
Figure 4:
Transient expression in rat adipose cells. A, the indicated reporter plasmids were transiently expressed
in primary rat adipose cells as described under ``Materials and
Methods.'' Reporter constructs contained obese ()
and luciferase (
) sequences as indicated and are shown to scale
except for the length of the luciferase. Results are luciferase
activity corrected for transfection efficiency and normalized to
p(-762)ob-luc = 100 and are the mean ± standard
error (number of independent experiments). Results <1 are not
clearly greater than the luminometer background. B, effect of
a C/EBP
expression vector on obese expression.
Transfections contained 2 µg of p(-762)ob-luc, 1 µg of
RSV-cat, the indicated amount of FlagCMV80-C/EBP
expression
vector, and pUC18 filler to give a total of 8 µg of plasmid DNA.
The raw RSV-cat data (
) and the p(-762)ob-luc data
corrected for chloramphenicol acetyltransferase expression (
) are
expressed relative to no C/EBP
vector. Data are the mean of two
independent experiments, except for the 4 µg point which is the
mean ± standard error from five independent
experiments.
A
number of adipose and hepatic genes are known to be regulated in
response to hormones and metabolic
state(28, 29, 30) . For example, insulin
and/or high glucose stimulate transcription of the genes for
glyceraldehyde-3-phosphate dehydrogenase(31) , fatty acid
synthase (32) , S(33) , stearoyl-CoA
desaturase I(34) , and pyruvate kinase and decreases the
transcription of the phosphoenolpyruvate carboxykinase(35) .
Recent evidence suggests that obese people have slightly elevated obese mRNA levels (36) . The transcription factor
binding motifs in the obese promoter, such as the putative
C/EBP site, may play a role in mediating this regulation.
Given the
postulated role of the obese gene as an adiposity sensor,
transcription of the obese gene may be sensitive to lipid
status. For example, polyunsaturated fatty acids decrease transcription
of hepatic pyruvate kinase (37) and fatty acid synthase (38) . With varying precision and certainty, candidate cis regulatory elements and their cognate trans factors have
been identified as mediators of these events. Two different direct
mechanisms are known to mediate the transcriptional response to
intracellular hydrophobic ligands. The sterol regulatory
element-binding proteins remain membrane-bound under sterol-replete
conditions but are proteolytically cleaved to release an active
transcription factor under low sterol conditions(39) . Ligand
binding to steroid hormone superfamily receptors causes the receptors
to bind DNA and activate transcription. In particular, peroxisome
proliferator-activated receptor is known to increase
transcription in response to linoleic acid, clofibric acid, and
thiazolidinediones (40, 41) and has been proposed to
regulate obese(40) . The mouse obese promoter
does not contain sequences that are identical to the reported sterol
regulatory element or peroxisome proliferator-activated receptor
regulatory element. However, obese may be controlled
indirectly by these factors, or directly via distant elements and/or
elements that do not conform to the classical sequence motifs.
Elucidation of the regulatory mechanisms controlling expression of the obese gene and of the promoter elements that confer adipose
specific expression will be important for understanding the regulation
of body fat in the normal state and the pathogenesis of obesity.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36238 [GenBank]and U36239[GenBank].