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Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892-1829
Address all correspondence and requests for reprints to: Simeon I. Taylor, Diabetes Branch, NIDDK, Bldg. 10, Room 9S213, 10 Center Drive MSC 1829, Bethesda MD 20892-1770. E-mail: sit{at}box-s.nih.gov
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, insulin treatment acutely increased both secretion and production of leptin by rat adipose tissue fragments. Isolated adipose cells lost most of their leptin during preparation, but they also showed the stimulatory effects of insulin. Immediately after isolation, leptin was seen in the endoplasmic reticulum (ER) of isolated adipose cells. After 10 min of insulin stimulation, the intensity of leptin immunostaining was reduced, which is consistent with increased secretion. Preliminary results have been presented previously (14, 15).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male rats (210–250 g; CD strain; Charles River Breeding
Laboratory, Wilmington, MA) were housed with free access to laboratory
chow and water. Animals were anesthetized using
CO2/O2 (70%:30%) and killed by decapitation
between 0009 h and 1100 h. All animals had food in their
stomachs indicating they had eaten during the previous dark cycle.
Animals were maintained in accordance with the NIH Guide for the Care
and Use of Laboratory Animals.
Tissue and cell isolation
To obtain tissue samples, epididymal fat pads were dissected
into proximal and distal segments during removal from the animal. These
segments were further sectioned into three pieces in
Krebs-Ringer-bicarbonate-HEPES buffer (KRBH) (10 mM
NaHCO3, 200 nM adenosine, 30 mM
HEPES, pH 7.4) containing 1% BSA (Fraction V) and 0.5 mg/ml
bacitracin. Each experimental sample consisted of one proximal and one
distal piece. This procedure ensured that each sample contained a
mixture of large and small cells, as leptin levels appear to be
correlated with the size of the adipose cells (data not shown and 16 . Six samples were obtained from a single animal; lipid content was
measured in one sample, a second sample was used to determine the
leptin content in freshly isolated tissue, and the remaining four
samples were used to determine the change in leptin levels as a
function of time. Two of these were incubated in the presence and two
in the absence of insulin. Thus, insulin-treated tissue was always
compared with untreated tissue from the same animal. After tissue
samples were blotted and weighed, one sample was placed immediately
into extraction buffer (1% Triton X-100, 0.3 M NaCl, 1
mM EDTA, 0.05 M Tris, pH 7.4) with complete
protease inhibitor cocktail (one tablet/50 ml, Boehringer Mannheim,
Indianapolis, IN), while the remaining samples were incubated at 37 C
in 2 ml KRBH buffer with 5% BSA and bacitracin, with or without 700
nM insulin (Eli Lilly, Indianapolis, IN). We chose this
insulin concentration, which is above the dose needed for maximal
stimulation of leptin secretion (data not shown and 8 for two
reasons: 1) to ensure high receptor occupancy quickly in tissue
fragments where some time might be needed for diffusion of insulin; and
2) to ensure maximal stimulation throughout a 4-h time course. At the
indicated times, the tissue was removed, blotted again, and homogenized
in 2 ml extraction buffer with a Dounce homogenizer (12 strokes with
loose pestle, followed by 12 strokes with tight pestle). Cell debris
was removed by centrifugation at 16,000 x g for 60 min
at 4 C.
For preparation of cells, epididymal fat pads were removed, minced, and digested with Type I collagenase as described previously (17). Three separate samples were analyzed for each time point during each experiment; the cells were incubated at a concentration of 15% (wt/vol) in 1.5 ml KRBH buffer with 5% BSA and bacitracin, separated by spinning through oil, and extracted as described for tissue.
Leptin antibody
Polyclonal antileptin antibodies were obtained by immunizing
rabbits (CYT Immune Services; College Park, MD) with 250 µg
glutathione S-transferase (GST)-murine leptin fusion protein
produced in Escherichia coli. Affinity-purified antibody was
produced by passing the serum over a GST-Sepharose column to remove
anti-GST antibodies and then passing the unbound material over a
GST-leptin column. Antileptin antibodies were eluted with isoglycine
buffer (0.1 M isoglycine, 0.1 M NaCl, pH
3).
Immunoprecipitation and immunoblotting
The cells or tissue extracts and untreated media from each
incubation were split into two samples that were immunoprecipitated and
quantified separately. Identical samples varied by 
20%, as did
different tissue samples from the same animal. For each sample, 25 µl
Ultra Plus Protein A beads (Pierce Chemical Co., Rockford, IL) were
washed three times in immunoprecipitation buffer (IB, 0.5% Triton
X-100, 0.3 M NaCl, 25 mM NaPO4, pH
7.4, 0.02% azide) containing 1% BSA (IB-BSA) and incubated at 4 C in
IB-BSA with 5 µl antileptin antiserum. After 4–6 h, the beads were
washed again and added to extracts or media samples and incubated
overnight at 4 C. The beads were sedimented by centrifugation and
washed once in IB-BSA, once in IB, and once in Tris-buffered saline, pH
7.4. The immunoprecipitates were solubilized and reduced by boiling in
Laemmli sample buffer containing 3.6% (wt/vol) SDS with 20
mM dithiothreitol. Our antimurine leptin antibody
quantitatively immunoprecipitated rat leptin. The solubilized
polypeptides were separated on 12.5% SDS-PAGE gels and transferred to
nitrocellulose by standard methods (18). Leptin was detected by
immunostaining with affinity-purified antimurine leptin antibodies
followed by goat antirabbit Fc-specific IgG coupled to horseradish
peroxidase (Jackson Immunolabs, West Grove, PA). The resulting bands
were visualized using enhanced chemiluminescence substrate (Pierce
Chemical Co.). Band intensities were quantified on a Molecular Dynamics
computing densitometer (Sunnyvale, CA). The relative amount of leptin
was determined by comparing the integrated density of experimental
bands to the integrated density of bands from immunoprecipitated
recombinant leptin standards, obtained by Factor Xa digestion of
GST-leptin fusion protein. After digestion with enzyme, the proteins
were separated by HPLC, and the fractions containing leptin were
combined. Our quantification consistently gave values 2- to 3-fold
lower than the same samples analyzed in the Linco rat leptin RIA (St.
Charles, MO). This discrepancy could be caused by a lower affinity of
our antibody for rat samples compared with the mouse standards;
however, this does not alter any of the conclusions based on
comparisons of the relative amounts of leptin in different samples. The
secretory rate was calculated as the quantity of leptin secreted into
the medium per gram wet weight of tissue divided by the length of time
the tissue was incubated.
Students’ t test was performed on Sigma Plot (Jandel Scientific Software, San Rafael, CA). Differences were accepted as significant at the P < 0.05 level.
Preparation and incubation of adipose cells for confocal
microscopy
White adipose cells were isolated as described previously (17).
At first we used a 1-h collagenase digestion and observed considerable
variation in the brightness of leptin immunofluorescence. In fact, some
freshly isolated cells had no detectable leptin staining. Shortening
the digestion time to 35 min increased the number of cells with bright
leptin immunostaining. This shorter digestion was used in the
experiments shown in
Figs. 6–9
. Isolated cells (2–4 x
106 cells/ml) were incubated without or with 700
nM insulin at 37 C for 15 min in KRBH with 1% BSA.
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Fluorescein isothiocyanate (FITC)-and lissamine rhodamine sulfonyl chloride-conjugated IgG, monovalent F(ab) fragments, and divalent F(ab')2 fragments from Jackson were the secondary antibodies. Specific antirabbit Ig antibodies were used with polyclonal primary antibodies, whereas antimouse Ig antibodies were used with monoclonal primary antibodies.
Indirect immunofluorescence microscopy
Single and double immunofluorescence experiments were performed
using adipose cells in suspension following the protocol described in
detail elsewhere (24). In some experiments, double-labeling
immunofluorescence was carried out with two polyclonal antibodies (25, 26). In these cases, the cells were first labeled with the polyclonal
anticalnexin antibody for 2 h at room temperature followed by an
affinity-purified FITC-conjugated goat F(ab) antirabbit IgG (heavy and
light chains) for 1 h at room temperature, following
manufacturer’s instructions (Jackson ImmunoResearch) so that all the
primary antibodies were sterically covered by the Fab fragments. Then,
the cells were incubated with the second polyclonal, antileptin
antibody for 2 h at room temperature, followed by a polyclonal
affinity-purified lissamine rhodamine sulfonyl
chloride-conjugated donkey F(ab')2 antirabbit IgG
(heavy and light chains) for 1 h at room temperature. Two control
experiments confirmed the effectiveness of this approach. First, no
reaction could be detected with a control, unconjugated polyclonal
affinity-purified goat F(ab) antirabbit IgG (heavy and light chains),
proving the efficacy of the monovalent F(ab) fragment. Second, the
pattern of the double labeled cells is identical to immunostaining with
each of the respective polyclonal antibodies alone. In all experiments,
the immunostained cells were viewed with a Nikon Optiphot 2
fluorescence microscope (Nikon Inc, Melville, NY) equipped with a
Bio-Rad MRC1000 or 1024 confocal laser scanning imaging system from
Bio-Rad Labs (Hercules, CA). This system uses a mixed argon/krypton
laser (
1 = 488 nm, blue line for FITC; 2
= 568 nm, yellow line for rhodamine) and COSMOS/LaserSharp (Bio-Rad
Labs, Hercules, CA) image analysis software. Specimens were viewed
using planapochromat x10/1,4NA, x60/1,4NA, and x100/1,4NA objectives.
For each experimental condition, 8–10 images per cell were recorded
from at least 10–15 cells. Images were collected sequentially for the
two fluorochromes in the double-labeling experiments and averaged with
a Kalman filter at an optical zoom setting of 1 to 2.5. Colocalization
was assessed throughout the cell by examination of several merged
images. For presentation, images were further enhanced digitally using
Adobe Photoshop 3.0 from Adobe Systems (Mountain View, CA) and printed
with a Kodak PS 8650 digital printer (Eastman Kodak, New Haven,
CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2A). This increase was significant
at all times studied (P < 0.05). After 10 min of
incubation, the amount of leptin remaining in insulin-treated tissue
was less than the amount in untreated tissue. After a 4-h incubation
without insulin, the amount of leptin in the tissue extracts decreased
to about 50% of the starting amount. However, insulin-stimulated
tissue retained 70–80% of the starting amount of leptin (Figs. 1B and 2B). Thus, at all times later than 10 min, insulin increased the total
amount of leptin produced, calculated by adding the amount of leptin in
tissue to the amount in the medium (Fig 2C). The increases in tissue-associated
leptin and total leptin were significant at all times greater than 10
min (P < 0.05).
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). Similar variability in leptin levels
was seen in isolated rat adipose cells. However, fresh tissue contained
4-fold more leptin per gram wet weight than an equivalent weight of
freshly isolated rat adipose cells.
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). However, in isolated cells, insulin
stimulation of leptin secretion was maintained for only 2 h (Fig. 4A). In contrast, insulin increased leptin secretion from tissue
fragments throughout the 4-h time course. Insulin-treated cells also
contained more leptin than untreated cells (Fig. 4B), although the
actual amount of leptin in all adipose cells decreased during the
in vitro incubation, as shown for tissue in Fig. 2
. Thus
insulin-treated cells produced more leptin during the first 2 h of
the in vitro incubation (Fig. 4C).
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). In cells with
detectable staining, leptin immunostaining was seen in a honeycomb
pattern, typical of proteins in the ER of adipose cells (Fig. 5, A-C).
This compartment is very distinctive in adipose cells. Previous
electron microscopy studies showed highly organized interconnected ER
membranes closely associated with the central large lipid droplet and
the small cytoplasmic lipid droplets; in fact, ER cisternae often
encase the lipid droplets (27, 28, 29). Because of this organization, at
the light level ER proteins display an immunofluorescence pattern that
outlines the lipid droplets. After 15 min of insulin treatment, two
patterns of leptin staining were found (Fig. 5, D-F). In many cells,
the staining was faint and restricted to a narrow rim near the plasma
membrane (Fig. 5D). In other cells, the leptin staining was bright, and
the outlines of the lipid droplets were still visible, indicating an ER
localization (Fig. 5, E and F). After 45 min of insulin treatment, more
cells showed bright staining, particularly in the presence of insulin,
perhaps due to the stimulation of de novo synthesis (data
not shown).
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, A-C). In cells not treated with
insulin, leptin staining was almost identical to calnexin staining.
This colocalization was seen in all focal planes throughout the adipose
cells. In cells where only dim leptin staining remained after insulin
treatment, most of the calnexin was present in regions that no longer
stained for leptin (Fig. 6, D-F). Leptin was still visualized in some
parts of the ER, possibly places in which leptin was being synthesized.
Leptin was never seen in a punctate staining pattern indicative of
storage in secretory vesicles, and leptin immunostaining did not
resemble the pattern of TGN 38 in the Golgi complex (data not
shown).
Variability in the amount of leptin was further documented by
immunofluorescence in both basal and insulin-treated cells. When whole
cells were fixed and stained immediately after isolation, fluorescent
signal was present in all cells, although the intensity of the staining
was variable (Fig. 7A). Generally, large
cells displayed brighter staining than small cells, which is consistent
with biochemical evidence that the former express higher levels of
leptin than the latter (data not shown and 16 . When cells were
incubated for 15 min without insulin, the fluorescence intensity
decreased, consistent with constitutive secretion of leptin (Fig. 7B).
Nonetheless, all the cells still exhibited detectable leptin staining.
In contrast, cells that were incubated in the presence of insulin for
15 min showed a greater decrease in overall fluorescence, and about
20% of the cells (74/380) no longer had any detectable leptin staining
(illustrated in the left panels of Fig. 8). We then investigated whether this
feature is specific for leptin by examining the fate of the ER-resident
protein calnexin. Figure 8 shows a montage of adipose cells treated
with insulin for 15 min and double stained for leptin and calnexin.
While the intensity of calnexin staining was relatively uniform (Fig. 8, D–F), the intensity of leptin staining was quite variable (Fig. 8, A–C). In cells with bright leptin staining, leptin staining matched
calnexin staining. However, in many cells the leptin staining was faint
or undetectable. Apparently these cells secreted their leptin in
response to insulin.
The immunostaining of leptin is different from the punctate
immunostaining of the GLUT4 glucose transporter (Fig. 9). In basal cells (incubated in the
absence of insulin), leptin and GLUT4 did not colocalize (Fig. 9, A and
B). Both proteins moved in response to insulin, but little overlap was
observed in the two distributions (Fig. 9, C and D). The focal plane
for these images was set at the cell surface where a secreted protein
such as leptin would not be expected to be found. Presumably, the
leptin staining came from protein on the way to the cell surface,
whereas the GLUT4 staining originates mainly at the plasma
membrane.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 3T3-L1 and 3T3-F422A adipocytes, it has been consistently reported that insulin increases the amount of leptin mRNA and the rate of leptin secretion (6, 7). However, it is not clear whether insulin exerts a specific effect to regulate synthesis and secretion of leptin or merely promotes adipocyte differentiation. In contrast, studies on insulin treatment of isolated human adipose cells have generally not shown acute stimulation of leptin secretion (12, 30). Studies in isolated rat adipose cells are equivocal: two studies reported effects at both the mRNA and protein levels while a third study showed no increase in leptin mRNA in response to incubation with insulin (4, 8, 10). Our results document stimulation of both production and secretion of leptin in tissue fragments. Moreover, these effects are easier to quantify in tissue fragments because isolated adipose cells contain much less leptin, thus making it difficult to see an effect. Furthermore, after 2 h in culture, isolated adipose cells no longer respond to insulin with increased leptin secretion, indicating they can easily lose the ability to produce leptin.
We observed that insulin increased leptin secretion in vitro. Why then have no increases in circulating leptin been detected in in vivo insulin clamp studies (12, 13, 31, 32, 33)? Possible simple explanations include a species difference between rats and humans or the possibility that some adipose cells do not respond like those in epididymal fat tissue. It could be that a delay occurs between secretion by fat cells and appearance in the blood. Another possibility is that insulin affects the clearance of leptin even though leptin appears to be cleared by passive filtration (34). However, the most likely explanation is that the direct effects of insulin are blunted by other effects in vivo. For example, in isolated rat adipose cells, the stimulation of leptin secretion by insulin is inhibited by stimulation of ß3-adrenergic receptors (8). Establishing which is the correct explanation is a fruitful area for further study.
Evidently, leptin is not stored in adipose cells or cell lines in culture, but rather is secreted as it is produced, leading to the suggestion that leptin secretion is regulated only by changes in transcription or translation (5, 6, 7). However, we observed a rapid increase in the rate of leptin secretion, even at times when the leptin content actually decreased. In addition, our morphological data suggest that insulin accelerated the movement of leptin out of the cell. Therefore, although leptin is not targeted to a stored pool of secretory vesicles, the secretion of leptin appears to be modulated by insulin. Insulin also stimulates secretion of adipsin in cultured adipocytes (35, 36). It is possible that insulin increases the movement of cargo throughout the secretory pathway, although it is not known whether leptin and adipsin are in the same transport vesicles after insulin treatment. We provide here the first evidence that, at this level of resolution, leptin is seen primarily in the ER as it is colocalized with a well established ER marker, calnexin (37, 38). Recent immunohistological studies of leptin localization are consistent with this localization, although the results from these studies have been interpreted as cytoplasmic staining (39, 40, 41). This discrepancy is probably technical in origin, possibly caused by the diffusion of the chromogen (39) and the low resolution of conventional immunofluorescence (40). Moreover, secreted proteins generally are cotranslationally inserted into the ER and then travel in vesicles to the cell surface. In 3T3-L1 adipocytes, leptin secretion is inhibited by brefeldin A, indicating that it is in this classical secretory pathway (5). Nonetheless, leptin staining within the Golgi or in secretory vesicles has not been detected. Although direct transport of cholesterol from the ER to the plasma membrane has been reported (42) and the integral membrane protein caveolin may also use this direct route (43), no examples are known of secretory proteins bypassing the Golgi complex on the way to the cell surface. It is tempting to speculate that leptin may be moving via a novel secretory pathway. However, it is more likely that because leptin is not glycosylated, it passes through the Golgi complex quickly without being concentrated there. Clearly, insulin affects leptin secretion from adipose cells by a mechanism that is distinct from the release of stored secretory vesicles.
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Acknowledgments |
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Received March 12, 1997.
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