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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 7 2550-2556
Copyright © 1999 by The Endocrine Society

Original Studies

Intrinsic Site-Specific Differences in the Expression of Leptin in Human Adipocytes and Its Autocrine Effects on Glucose Uptake1

Hui H. Zhang, Sudhesh Kumar, Anthony H. Barnett and Margaret C. Eggo

Division of Medical Sciences, University of Birmingham, Birmingham, B15 2TH, United Kingdom

Address all correspondence and requests for reprints to: Dr. Margaret C. Eggo, Department of Medicine, Queen Elizabeth Hospital, Birmingham, B15 2TH, United Kingdom. E-mail: eggomc{at}novell5.bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin, the ob gene product of adipocytes, regulates body weight by actions on the satiety center in the hypothalamus, but it may also have peripheral effects on the metabolic actions of insulin. In human mature adipocytes isolated from omental (OM) and sc tissue, we found that leptin (10 and 100 ng/mL) significantly reduced insulin-mediated glucose uptake by 40% (P < 0.05). The effects were rapid and sustained. A U-shaped dose-response curve was obtained, and high leptin concentrations (>100 ng/mL) were without effect. Leptin did not affect basal glucose uptake in adipocytes and had no effect on insulin-stimulated glucose uptake in human preadipocytes. Because leptin may thus have autocrine effects, we examined leptin production from OM and sc adipocytes. Western blotting of leptin from 96-h conditioned medium showed greater leptin secretion from sc than OM adipocytes, with a ratio of 3.2 (SE ± 0.3, P < 0.01). Long-term ceiling cultures were used to examine intrinsic differences in leptin expression under closely controlled conditions. Confocal immunofluorescence microscopy of 12- to 16-day-old ceiling-cultured adipocytes showed that sc adipocytes contained 3.4-fold more leptin (SE ± 0.5, P < 0.01) than OM adipocytes, indicating an intrinsic site-specific difference in leptin production. The autocrine effects of leptin to inhibit insulin-stimulated glucose uptake and subsequent lipogenesis in adipose tissue may, therefore, be less in OM adipocytes and may play a role in determining visceral obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROENDOCRINE effects of leptin and its role in regulating body weight are now well established. In humans, mutations in leptin (1, 2) or its receptor (3) result in morbid obesity and follow findings in ob/ob and db/db mice, where leptin deficiency caused by mutations in the ob gene or leptin resistance caused by mutations in the db gene (leptin receptor) result in extreme obesity (4, 5, 6). Administration of recombinant leptin in ob/ob mice inhibits synthesis and release of hypothalamic neuropeptide Y and subsequently reverses the obese phenotype, indicating that leptin functions in a negative feedback loop, from adipose tissue to the satiety centre in the hypothalamus, to regulate body adiposity (4, 7, 8).

The common form of human obesity, however, is associated with high circulating levels of leptin that correlate with body mass index (9, 10). Because leptin is produced primarily by adipocytes, this increase in circulating leptin could reflect the increased adipose mass, leptin resistance, and/or an attempt to counterbalance the increase in body adiposity. Elevated plasma leptin levels are also associated with insulin resistance, independent of body adiposity (11, 12, 13); and the insulin action enhancer, troglitazone, down-regulates leptin expression and improves insulin sensitivity (14, 15). These data suggest that leptin may exert cellular effects on insulin-sensitive, peripheral target tissues. Leptin receptors are present in adipose tissue where long and short forms are expressed, suggesting potential autocrine effects (16, 17).

Studies of the direct effects of leptin on adipose tissues have yielded controversial data. Using the mouse 3T3-L1 preadipocyte cell line and primary cultures of rat adipocytes, several groups have failed to show any effects of leptin on glucose transport in short- or long-term studies (18, 19, 20). However, earlier studies, also using rat adipocytes, showed acute effects of leptin on insulin binding (21); and longer-term studies have shown inhibitory effects of leptin on insulin action on glucose transport, glycogen synthase, lipogenesis, lipolysis, and protein synthesis (22, 23, 24). In retroperitoneal fat pads, lipolysis was increased by leptin; and in human hepatic cells, which also express the leptin receptor, leptin attenuated insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 but increased phosphatidylinositol 3-kinase activity (25). There have been no studies using human adipocytes to examine the potential autocrine role of leptin on insulin actions in these cells. We have examined the effect of leptin on insulin-stimulated glucose uptake in human omental (OM) and sc adipocytes. We examined the effects in adipocytes from two different fat depots, because of the recognized association of central (visceral) obesity with insulin-resistance and the metabolic syndrome.

Leptin is produced by both human OM and sc adipocytes. Studies have shown that leptin messenger RNA (mRNA) expression was lower in freshly isolated OM adipocytes than in sc adipocytes (26, 27, 28). Leptin secretion, during a 2-h incubation, has also been shown to be less from OM than from sc adipose tissue explants (29). The site-specific differences in leptin production may indicate the differential contribution of OM and sc adipocytes, in providing leptin for the regulation of body weight, which is mediated via its central neuroendocrine and putative peripheral effects. Whether the differences observed in these studies are attributable to in vivo site-specific differences in adipocyte exposure to regulators of leptin production or to the intrinsic properties of adipocytes themselves has not been addressed. To determine whether there is an intrinsic site-specific difference in leptin production in OM and sc adipocytes, we compared leptin secretion from isolated human OM and sc adipocytes (by Western blotting of 96-h conditioned medium) and leptin protein expression per cell (by confocal immunofluorescence microscopy of leptin in long-term, ceiling-cultured OM and sc adipocytes).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of human adipocytes and preadipocytes

OM and sc adipose tissue were obtained from 12 patients undergoing elective abdominal surgery in accordance with guidelines of the local ethical committee. Eight women (age, 33–72 yr; BW, 67.0–80.7 kg) and 4 men (age, 45–68 yr; BW, 74.2–85.7 kg) were fasted for at least 6 h preoperatively, and all underwent general anesthesia. None of the patients had diabetes or severe systemic illness, and none were on medications known to influence adipose tissue mass, distribution, or metabolism. Paired OM and sc tissue from the same patients (n = 3) was used for each of the 4 experimental protocols, including the dose effect of leptin on glucose uptake, the time course of the effect, leptin secretion, and leptin protein expression. Adipocytes were isolated by the method of Rodbell (30), with modifications. At no time in the isolation procedure were adipocytes cooled below room temperature. The tissue was washed with HBSS (Gibco BRL Life Technologies, Paisley, UK), visible blood vessels were removed, and the tissue was cut with scalpel blades. Freshly diced tissue pieces were digested with type I collagenase (1 mg/mL in HBSS, Worthington Biochemical Corp., Freehold, NJ) for 1 h in 37 C water bath, shaken at 100 cycles/min. The disrupted tissue was filtered through a double-layered cotton mesh, and adipocytes were washed 3 times with DMEM:F12 medium (Gibco BRL Life Technologies) and centrifuged at 250 x g for 5 min. The mature adipocytes at the surface, after centrifugation, were either cultured, short term, in suspension culture in DMEM-F12 medium or in long-term ceiling cultures (see later). The preadipocytes in the pellets were resuspended in red blood cell lysing buffer (154 mmol/L NH4Cl, 5.7 mmol/L K2HPO4, 0.1 mmol/L EDTA) and incubated for 10 min. Cells were centrifuged at 150 x g for 5 min and resuspended in DMEM:F12 medium supplemented with 15% bovine FCS (First Link UK Ltd., Brierley Hill, UK). After overnight incubation, cells were incubated in serum-free DMEM:F12, containing 10 µg/mL transferrin (Sigma Chemical Co., Poole, UK), for 24 h.

Glucose uptake experiments

Triplicate samples of isolated OM and sc adipocytes were cultured in suspension with varying concentrations of recombinant human leptin (0–10 µg/mL, R&D Systems Europe Ltd, Abington, UK) at 37 C in a 5% CO2 incubator for 24 h. Glucose uptake in adipocytes was measured by determining the uptake of 14C-glucose at trace concentrations, as described previously (31, 32). The method has been validated, by these investigators, for measuring glucose uptake in comparison with 3-O-methyl glucose transport in human adipocytes. With this method, glucose metabolites do not escape from the cells in detectable quantity, and more than 99% of the radioactivity remains in the incubation medium as glucose. Briefly, adipocytes (treated with/without leptin) were incubated with 300 nmol/L 14C-glucose (313 mCi/mmol, ICN Pharmaceuticals Ltd, Basingstoke, UK), in the presence/absence of 100 nmol/L insulin (Sigma Chemical Co.), for 60 min at 37 C in a 5% CO2 atmosphere, in 10 mmol/L HEPES in Krebs Ringer Buffer (KRB; pH 7.4) containing 3% BSA (First Link UK Ltd.). The cells were separated by centrifuging through silicone oil at 10,000 x g for 30 sec. Cellular DNA content was quantified by a fluorochrome assay described later in the text. 14C-radioactivity associated with the cells was determined by liquid scintillation counting and corrected for cellular DNA content. Time-course experiments were performed by preincubating OM and sc adipocytes, with/without recombinant human leptin (100 ng/mL), for the indicated periods of time (0–72 h).

In preadipocytes glucose, uptake was determined after 48 h incubation in serum-free medium. Triplicate samples in 24-well 2-cm2 plates were incubated with indicated concentrations of recombinant leptin for 24 h, followed by 1 h incubation in KRB buffer containing 300 nmol/L 14C-glucose. The cell layer was washed briefly three times with KRB buffer. Cellular DNA was quantified using the fluorochrome assay.

The fluorochrome assay for adipocyte DNA quantification

Quantitation of DNA in adipocytes and adipose stromal cells was performed using a fluorochrome assay method, as previously described, with modifications (33, 34). Hoechst 33342 (Sigma Chemical Co.), used at a concentration of 0.5 µg/mL in phosphate saline buffer (0.05 mol/L sodium phosphate, 2 mol/L NaCl, pH 7.4), was added to adipocytes. After 15 min incubation in the dark, fluorescence was measured on a fluorometer (Perkin-Elmer Corp., Beaconsfield, UK). A 356-nm excitation filter and a 458-nm emission filter were used.

Western blotting of leptin secreted by OM and sc adipocytes

OM and sc adipocytes (2 x 106) were cultured for 96 h in suspension in 10 mL DMEM:F12 medium, with/without 100 nmol/L insulin (Sigma Chemical Co.) and 100 nmol/L dexamethasone (Sigma Chemical Co.). Secreted proteins from the same number of cells were precipitated with 3 vol absolute ethanol, overnight at -20 C. After centrifugation, the pellets were dissolved in 80 µL sample buffer, and proteins were separated on a 15% SDS polyacrylamide gel with a 7.5% stack, using Tris-glycine buffers (35). Prestained molecular weight markers (Sigma Chemical Co.) and recombinant human leptin (R&D Systems Europe Ltd.) were also analyzed. The separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (ICN Pharmaceuticals Ltd.). Nonspecific binding was blocked by incubating the membrane in 10% nonfat milk in Tris-buffered saline (TBS, pH 7.4) for 2 h at room temperature. The membranes were incubated with primary antibody, raised in sheep against the N-terminal amino acids of leptin (The Binding Site, Birmingham, UK), for 1 h, at a dilution of 1:500 in TBS with 0.1% BSA. After exposure to primary antibody, the blots were washed in TBS-Tween 20 and exposed to a 1:40,000 dilution of rabbit antisheep antibody (Calbiochem, Nottingham, UK) in TBS-Tween 20 with 3% nonfat milk, for 1 h. The blots were detected by the enhanced chemiluminescence system (ECL, Amersham Life Sciences Ltd, Little Chalfont, UK) and were analyzed using Gel blot software [Gel Analysis Suite for Windows (c) 93].

Immunofluorescence staining of leptin in ceiling-cultured adipocytes

To verify findings from the leptin secretion study, we examined cellular leptin protein expression in 12- to 16-day-old human OM and sc adipocytes cultured using a modified ceiling culture method that was first described by Sugihara et al. (36). Isolated OM and sc adipocytes (50 µL of packed adipocytes circa 105 cells) were inoculated into 2 mL DMEM:F12 with 20% new born bovine calf serum in a 6-well plate (9.6 cm2, Nalge Nunc International, Hereford, UK). The cells were covered with a sterile 20-mm x 20-mm glass coverslip, and adipocytes attached to the underside of the coverslip. Ten- to 14-day-old OM and sc adipocytes, attached to the ceiling surface, were washed with HBSS and incubated in DMEM:F12 for 48 h. After incubation, the coverslips were air-dried for 1 h. Cells were fixed with cold acetone (Fisher Scientific UK Ltd., Loughborough, UK) for 5 min, washed twice with PBS (120 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L sodium phosphate, pH 7.6, at 25 C) for 5 min and blocked with 10% BSA in PBS at room temperature for 30 min. After removing excess blocking solution, cells were incubated with antileptin IgG ([0.3 mg/mL in PBS], The Binding Site) for 30 min. Primary antibodies were omitted for negative control samples. The cells were washed with PBS twice for 5 min and further incubated with fluorescein isothiocyanate (FITC)-conjugated donkey antisheep IgG (1:50 dilution with PBS, The Binding Site) for 30 min. After washing with PBS twice for 5–10 min, cell nuclei were stained with propidium iodide (0.25 µg/mL in PBS, Sigma Chemical Co.) for 30 sec and washed with PBS twice for 5 min. After brief drying to remove excess PBS solution, 2.5% 1,4-diazabicyclo-[2.2.2] octane (Sigma Chemical Co.) in 80% glycerol was added to stained cells to inhibit fading of fluorescence (37). Staining of leptin was viewed by confocal immunofluorescence microscopy.

Confocal laser scanning microscopy of leptin staining

Cellular leptin expression was analyzed by using a Bio-Rad MRC 500 confocal microscope (Bio-Rad Laboratories, Inc., Richmond, CA), which allows measurements of total cell area, integrated fluorescence intensity, and subcellular distribution of leptin immunofluorescence. To compare the relative amounts of leptin in OM and sc adipocytes, 30 images were collected at a standard magnification. The integrated fluorescence intensity (mean intensity x area) was recorded for each cell image. Values from negative control samples were subtracted from the test values. This was performed on 3 cell culture preparations from different patients of paired tissue from sc and OM sites. The integrated and mean fluorescence intensity of OM and sc adipocytes were compared.

Statistics

All experiments in the study were performed on adipocytes from at least three patients. Data from representative preparations are shown. One-way ANOVA was used for glucose uptake analysis. A paired Student’s t test was used for data analysis of leptin immunofluorescence in OM and sc adipocytes. Data are means ± SE or means ± SD. P values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of leptin on non-insulin-mediated glucose uptake in OM and sc adipocytes

To examine the effects of leptin on glucose uptake, OM and sc adipocytes were preincubated with varying concentrations of leptin for 24 h. Cell viability was checked by exclusion of trypan blue and was more than 80% at the end of primary cell culture. Glucose uptake assays were performed in the absence of insulin. Leptin preincubation did not have any significant effects on glucose uptake in adipocytes, as shown in Fig. 1Go.



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  		Figure 1. The effect of leptin on non-insulin-mediated glucose uptake in human OM (•) and sc ({circ}) adipocytes. Results from a representative experiment (n = 3) are shown.

 
Effects of leptin on insulin-mediated glucose uptake in mature adipocytes and preadipocytes

OM and sc adipocytes were incubated with increasing concentrations of leptin for 24 h, followed by glucose uptake assays in the presence/absence of 100 nmol/L insulin (Fig. 2AGo). Preincubation with leptin produced a U-shaped dose-response curve. Leptin, at concentrations of 10 and 100 ng/mL, significantly reduced insulin-stimulated glucose, by 40%, in both OM and sc adipocytes (P < 0.05). At lower (0.1 ng/mL and 1 ng/mL) and higher (1 µg/mL and 10 µg/mL) concentrations, leptin did not significantly decrease insulin-stimulated glucose uptake.



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  		Figure 2. A, The effects of varying concentrations of leptin (24-h incubation) on insulin-mediated glucose uptake (P < 0.05 at 10 ng/mL and 100 ng/mL); B, the time course of the effects of preincubation in 100 ng/mL leptin in OM (•) and sc ({circ}) adipocytes, P < 0.05 at all time points (0–72 h). The results of a representative experiment (n = 3) are shown.

 
To determine the short- and long-term effects of leptin, OM and sc adipocytes were preincubated with recombinant human leptin (100 ng/mL) for the indicated incubation periods before glucose uptake assay. Leptin significantly inhibited insulin-stimulated glucose uptake in OM and sc adipocytes at all time points. Simultaneous exposure to leptin and insulin during 60-min glucose uptake assay also significantly decreased glucose uptake in both cell types (Fig. 2BGo, P < 0.05).

The effect of 24-h leptin pretreatment (100 ng/mL) on the uptake of 14C-glucose in preadipocytes, in the presence of insulin, was determined as described for adipocytes. In contrast to its effects in adipocytes, leptin did not affect insulin-mediated glucose uptake (data not shown).

Western blotting of leptin secreted by OM and sc adipocytes

Western blots, probing for leptin in the conditioned medium from 96-h suspension cultures of adipocytes, are shown in Fig. 3Go, A and B. A single band was identified by the antileptin antibody at 16 kDa, which comigrated with the recombinant human leptin. In the absence of insulin and dexamethasone, leptin secretion was low from sc, and nondetectable from OM adipocytes. To quantitate the differences in leptin secretion, adipocytes were incubated with insulin and dexamethasone, which have been shown to increase leptin production and secretion (38, 39, 40). Densitometry indicated that sc adipocytes secreted 3.2-fold (SE ± 0.3, P < 0.01) leptin, compared with OM adipocytes, during the 96-h incubation. Cell viability was determined by trypan blue exclusion at the end of the 96-h incubation and, there was no significant interdepot difference in cell viability.



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  		Figure 3. Western blotting for leptin secreted by isolated human adipocytes in suspension culture over 96 h. A, Leptin secreted by adipocytes cultured in DMEM:F12; B, leptin secreted by adipocytes cultured in DMEM:F12 supplemented with 100 nmol/L insulin and 100 nmol/L dexamethasone. In both blots, lane 1 is the positive control, recombinant human leptin. Lane 2, leptin secreted by sc adipocytes; lane 3, leptin secreted by OM adipocytes.

 
Comparison of leptin expression in ceiling cultures of OM and sc adipocytes

Isolated adipocytes floated to the top of the medium in culture plates. During the first 2 days of ceiling culture, they adhered loosely to floating coverslips; and within 10–14 days, they became firmly adherent to the undersurface of the coverslip. Adipocytes maintained their unilocular morphology throughout the incubation period. Cell viability was checked by exclusion of trypan blue and was more than 85% at the end of 10- to 14-day incubation. No significant interdepot differences were detected.

Ceiling-cultured OM and sc adipocytes were stained with antileptin antibody and FITC-conjugated secondary antibody. Leptin was shown in both sc (Fig. 4AGo) and OM (Fig. 4BGo) adipocytes, indicated by the green fluorescence. Figure 4Go, C and D, represents the negative staining of sc and OM adipocytes treated only with the FITC-conjugated secondary antibody. Individual adipocytes were identified by the red fluorescence staining of cell nuclei with propidium iodide and their unilocular morphology, distinguishable from the fibroblast-like appearance of preadipocytes. Areas where propidium iodide staining and fluorescein staining were coincident appear yellow. Leptin staining in OM and sc adipocytes taken from the same patients (n = 3) was quantified by confocal microscopy, and data are shown in Tables 1Go and 2Go. Thirty images from each cover slip were examined, and the mean integrated fluorescent leptin staining (per cell) was determined. There was a 3.4-fold-higher level of leptin (SE ± 0.5, P < 0.01) in sc than in OM adipocytes (Table 1Go). OM adipocytes were about 30% smaller than sc adipocytes (P < 0.05). When the total cell area (cell size) of individual adipocytes was taken into account, the ratio was reduced to 2.1 (SE ± 0.1, P < 0.01, Table 2Go).



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  		Figure 4. Immunofluorescence staining of leptin in ceiling-cultured sc and OM adipocytes. Magnification, x400. A, Staining of leptin in sc adipocytes; B, staining of leptin in OM adipocytes; C and D, background controls showing staining in the absence of primary antibody.

 

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  		Table 1. The integrated fluorescence intensity of leptin staining (per cell, x10-6)

 

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  		Table 2. The mean fluorescence intensity of leptin staining (per cell area)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that recombinant human leptin significantly inhibited insulin-mediated glucose uptake in both OM and sc adipocytes. The dose effects produced a U-shaped curve. Inhibitory effects of leptin were evident at physiological concentrations that are in the range of 1–100 ng/mL (41). The lack of effects at high concentrations could be attributable to the negative cooperativity caused by the dimerization and oligomerization of leptin receptors at high leptin concentrations, because leptin receptors have been shown to exist in the form of monomer, dimer, and oligomer (42). The absence of effects at high concentrations indicates that the inhibitory effects observed at lower concentrations were not caused by any cytotoxic contaminants in the recombinant leptin. The U-shaped response may explain some of the disparities in the literature concerning the direct effects of leptin on adipocytes (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Other explanations for the absence of effect of leptin on glucose metabolism could be attributable to loss of leptin receptor during adipocyte isolation or to species differences. This is the only study to be performed on human adipocytes. The rapid effect of leptin on insulin-mediated glucose uptake suggests that leptin may affect early insulin signaling pathways, as observed in rat-1 fibroblasts (43), rat adipocytes (21), and human HepG2 cells (25).

The rate of glucose uptake is considered rate limiting for fatty acid synthesis and triglyceride storage in adipocytes under most conditions (44). The major intracellular metabolic pathway for glucose in human adipocytes has been shown to be lipogenesis, with 80% of glucose being converted to lipid and the remaining 20% being found in the water-soluble fraction (45). Leptin inhibited insulin-mediated glucose uptake in our study. We hypothesize that adipocyte leptin may have an autocrine effect on insulin-mediated glucose uptake, preventing excessive lipid storage. Our findings are compatible with data from other studies showing that leptin suppressed hormone-induced lipid synthesis in 30A5 preadipocytes (46) and inhibited insulin-mediated glucose uptake and glucose utilization in lipid synthesis, and increased lipolysis in rat adipocytes (20, 21, 22). The interactions between insulin and leptin actions are important, because insulin resistance has been postulated to protect against further weight gain (47), and leptin may also contribute to the insulin resistance.

Leptin did not produce significant effects on insulin-stimulated glucose uptake in preadipocytes. Compared with adipocytes, preadipocytes are relatively nonresponsive to physiological effects of insulin because they possess few insulin receptors and insulin-responsive glucose transporter 4, despite the possession of a large number of insulin-like growth factor I receptors that bind insulin with a 70-fold lower affinity than insulin-like growth factor I (48, 49). The number and affinity of insulin receptors increase approximately 25-fold during differentiation, after which adipocytes become exquisitely sensitive to insulin (48, 50). In addition, preadipocyte differentiation is also accompanied by the increase in the number of glucose transporter 4 transporters (51, 52). Therefore, the lack of effect could be caused by absence of either leptin or insulin receptors in preadipocytes.

To study leptin secretion, isolated adipocytes (rather than tissue explants) were used. OM tissue contains a higher proportion of vascular stromal cells than sc tissue, and these site-specific differences in tissue composition were thus controlled. In the absence of insulin and dexamethasone, leptin secretion from sc adipocytes was higher than from OM adipocytes, which was nondetectable by Western blotting. Insulin and glucocorticoids are increased in obese people and have been previously shown to increase leptin production (38, 39, 40). To induce and quantitate leptin secretion, adipocytes were cultured in the presence of insulin and dexamethasone. Western blotting of leptin from the conditioned medium showed 3.2-fold (SE ± 0.3) increased leptin secretion from sc, compared with OM adipocytes. Compared with sc adipocytes, OM adipocytes have been shown to express more glucocorticoid receptors (53, 54) but are more resistant to the antilipolytic action of insulin and are smaller (29, 55).

To determine whether the differences in leptin secretion are caused by intrinsic site-specific differences in leptin production, leptin protein expression per cell and per cell area was examined using 12- to 16-day-old ceiling-cultured adipocytes. We have used this method to study adipocyte apoptosis, proliferation, and tumor necrosis factor-{alpha} expression (56). In this study, we have (for the first time) visually observed subcellular protein expression of leptin in human adipocytes. Confocal microscopy showed 3.4-fold (SE ± 0.5, P < 0.01) leptin in sc adipocytes, compared with OM adipocytes. When the fluorescence intensity of leptin was corrected for cell size (integrated intensity/total cell area), the differences in leptin expression were reduced to a ratio of 2.1 ± 0.1 (P < 0.01), indicating that cell size and other adipocyte-specific factors may determine leptin production. OM and sc adipocytes have been shown to be intrinsically different in many of their properties, such as the differential expression of PAI-1 (57) and the control of lipolysis (58, 59). The differences in leptin production in 12- to 16-day-old OM and sc adipocytes may be caused by depot-specific differences in the intrinsic properties of adipocytes.

The lower production of leptin by OM (than sc) adipocytes suggests that visceral adipose tissue may be a less important contributor to circulating leptin and thereby plays a lesser role in providing the hormonal feedback for the control of body weight. At the local tissue level, we postulate that the lower production of leptin may lead to reduced inhibitory effect of leptin on insulin-mediated glucose uptake and subsequent glucose-derived lipid storage in OM adipocytes. This is consistent with the model recently described by Friedman and Halaas (60), who propose that low levels of leptin (below a set point) may result in expansion of adipose tissue mass and subsequent obesity. As discussed by Montague et al. (27) in their study of leptin mRNA levels in OM and sc tissue, in times of variable food supply, the intrinsic lower production of leptin by OM adipocytes may have a survival advantage, in allowing maximum storage of energy when food is available. However, it may be disadvantageous in Western societies, in which a plentiful food supply may predispose to visceral obesity and associated metabolic diseases.


   Acknowledgments
 
We thank Mr. Simon Bramhall and all the operative surgeons and theater staff at the University Hospitals Trust, who kindly provided fat samples from patients for the studies.


   Footnotes
 
1 This work was supported by the British Diabetic Association and Eli Lilly & Co. Industries, Basingstoke, UK. Antileptin antibody was kindly provided by Dr. A. C. Bradwell, of The Binding Site Ltd. and the University of Birmingham. Back

Received November 11, 1998.

Revised February 16, 1999.

Revised April 2, 1999.

Accepted April 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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