(Received for publication, September 12, 1995; and in revised form, November 7, 1995)
From the
The product of the recently cloned mouse obese (ob) gene is likely to play an important role in a loop regulating the size of the adipose tissue mass. The hormonal regulation of the ob gene could affect adiposity. To investigate this point, the effect of insulin on ob gene expression was examined in cells of the 3T3-F442A preadipocyte clonal line. ob mRNA is absent from exponentially growing, undifferentiated cells as well as from confluent preadipose cells. Terminal differentiation of preadipose to adipose cells leads to the expression of ob mRNA detected by a sensitive and quantitative ribonuclease protection assay. In adipose cells, the level of ob mRNA is sensitive to insulin in the nanomolar range of concentrations with an increase from an average of 1 copy to 5-10 copies/cell. The effect of insulin was fully reversible and takes place primarily at a transcriptional level. The ob mRNA shows a rapid turnover, with a half-life of approximately 2 h in the absence or presence of insulin. The level of secreted Ob protein is also regulated by insulin. These results indicate that the ob gene is expressed in mature fat cells only and support the possibility that insulin is an important regulator of ob gene expression.
The ``lipostat'' or ``adipostat'' theory
postulates that the size of body fat stores is regulated by a feedback
loop(1) . This hypothesis is based upon the recovery of initial
body weight following lipectomy (2) and parabiosis experiments
between genetically obese and wild-type mice suggesting the existence
of putative factor(s) regulating food intake(3) . The recently
cloned ob gene from mouse, rat, and human encodes a
circulating factor of 16 kDa that is secreted from adipocytes from
various adipose
depots(4, 5, 6, 7, 8) . The
OB protein, named leptin, appears to act at a distant site since
injections of the leptin decrease food intake and body weight in ob/ob
mice and their lean
counterparts(9, 10, 11, 12) . This
phenomenon implicates directly or indirectly the hypothalamus since
mice with chemical lesions of the ventromedial nucleus of the
hypothalamus (VMH), ()after becoming rapidly
hyperinsulinemic, express a dramatic increase in the levels of ob mRNA(5, 13) . A substantial fall in ob mRNA in the epididymal fat of lean mice has been observed after
fasting; this phenomenon is rapidly reversed on
refeeding(13, 14, 15, 16) . The
correlation between insulin level and the levels of ob mRNA
and plasma leptin suggests that insulin may have direct effects on ob gene
expression(15, 16, 17, 18) . In this
paper, we present data using cultured adipocytes that support this
hypothesis.
Figure 1:
Expression of ob mRNA during
differentiation of 3T3-F442A cells. Cells were maintained from
confluence in differentiation medium. Total RNAs were prepared at the
indicated times. ob transcript has been determined using a
ribonuclease protection assay. Expression of A2COL6 and adipsin
transcripts have been determined by performing Northern blot analysis.
An equivalent amount of intact RNA was run in each condition as
indicated by hybridization to a GAPDH probe and -actin probe (not
shown). The results are standardized to GAPDH mRNA signals and
expressed by taking as 100% the maximal signal obtained for each probe.
, A2COL6 mRNA;
, adipsin mRNA;
, ob mRNA.
Figure 2:
ob RNA content in 3T3-F442A
adipose cells after insulin removal followed by insulin addition. Left panel: a, confluent cells were maintained for 10
days in differentiation medium; b, cells maintained as in a were treated to remove insulin as described under
``Experimental Procedures'' and then exposed to standard
medium supplemented with 2 nM T for 24 h; c, insulin-deprived cells obtained as in b were
exposed to 3 nM insulin for 48 h. Total RNAs were prepared in
each condition, and determination of ob and adipsin RNAs
contents were performed as described in Fig. 1. Right
panel, the results are standardized to GAPDH mRNA signals and
expressed by taking as 100% the maximal signal obtained for each probe.
, ob mRNA;
, adipsin
mRNA.
Figure 3:
Time
course of insulin effect on ob RNA level in 3T3-F442A adipose
cells. After 10 days in differentiation medium adipose cells were
treated to remove insulin as described in Fig. 2. Then, 1 nM insulin (open symbols) or 3 nM insulin (black symbols) were added at time zero to standard medium
containing 2 nM T. At the indicated times, RNAs
were prepared and analyzed as described in Fig. 1. The results
are standardized to GAPDH mRNA signals and expressed by taking as 100%
the maximal signal obtained in each condition.
,
, ob mRNA;
, adipsin mRNA.
Figure 4: Dose-response relationship of insulin to ob RNA accumulation. Left panel, control cells (C) were maintained in differentiation medium for 10 days and analyzed for ob and GAPDH mRNA content using ribonuclease protection assays (6-day exposure time). In parallel experiments, control cells were treated to remove insulin (defined as condition 0) as described in Fig. 2, and insulin was added at various concentrations for 24 h. Probes (P) and protected fragments are indicated. Right panel, the results are standardized to GAPDH mRNA signals (12-h exposure time) and expressed by taking as 100% the stimulation observed at 3 nM insulin.
Figure 5: Secreted leptin in the absence or the presence of insulin. Twelve-day postconfluent, differentiated 3T3-F442A cells were maintained under serum-free conditions as described under ``Experimental Procedures.'' Leptin concentration was measured by immunoprecipitation in the presence or absence of insulin. Lane 1, 24-h protein expression in the presence of 3 nM insulin; lanes 2 and 4, expression after 24 and 48 h in the absence of insulin, respectively; lanes 3 and 5, 24-h expression after readdition of insulin (1 and 3 nM, respectively); lane 6, serum-free medium alone; lane 7, 24-h protein expression in the presence of 3 nM insulin in 2-day postconfluent, undifferentiated cells; lane 8, serum-containing medium alone.
Figure 6:
Half-life of ob mRNA and
transcriptional activation of ob gene by insulin in adipose
cells. Upper panel, postconfluent 3T3-F442A cells were
maintained in standard medium containing 2 nM T for 10 days and then supplemented (A) or not (B) with 10 nM insulin for 24 h (defined as time
zero). Actinomycin D (2 µg/ml) was added for 2 h. The results are
standardized to GAPDH mRNA signals (12-h exposure time) and expressed
by taking as 100% the signal obtained in each condition of culture at
time zero of the actinomycin treatment. Lower panel, cells
were maintained for 10 days in differentiation medium (a) or
in differentiation medium followed by insulin removal (b) as
described in Fig. 2. Then 1 or 3 nM insulin were added
for 15 h in the absence (c, d) or the presence (e, f) of 5 µg/ml actinomycin D. Analysis for ob and GAPDH mRNA using ribonuclease protection assays was
then performed (7-day exposure time).
In conclusion, ob gene is expressed in adipose cells only, and its regulation is reversibly and exquisitely sensitive to insulin at physiological concentrations. Secreted leptin can be detected in conditioned media of differentiated cells exposed to the hormone, and its concentration is also modulated by insulin in a reversible manner. The low expression of ob gene in vitro compared with in vivo suggests that other factors are required for maximal expression. The identification of additional factors that regulate ob gene expression is under investigation. It also appears that glucocorticoids regulate ob gene expression in vivo(26) , but the pharmacological doses used in this study prevent any definite conclusion. Adipogenic factors other than insulin, which have been reported to trigger terminal differentiation of adipose precursor cells to adipose cells(27) , are likely to play a regulatory role in the expression of ob gene.