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Endocrinology Vol. 142, No. 8 3558-3562
Copyright © 2001 by The Endocrine Society

ARTICLES

The Effects of Insulin, Glucose, and Pyruvate on the Kinetics of Leptin Secretion

James R. Levy and Wayne Stevens

Section of Endocrinology and Metabolism, McGuire Veterans Administration Medical Center (J.R.L.), and Medical College of Virginia/Virginia Commonwealth University (J.R.L.), Richmond, Virginia 23249


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin is a hormone that is secreted by fat cells and has roles in body weight regulation, glucose metabolism, reproduction, and other neuroendocrine functions. The purpose of this study was to determine whether the secretagogues, insulin, glucose, and pyruvate, enhance leptin secretion by increasing leptin synthesis, or whether these secretagogues stimulate the quantal release of a stored cytosolic pool of leptin. We found that in the absence of secretagogues, the rate of leptin secretion from isolated rat adipocytes approximately equals the rate of leptin synthesis. For 60 min after the addition of secretagogues, leptin synthesis rapidly increases, with little or no leptin secretion; leptin increases intracellularly by approximately 60% (P < 0.05). After 60 min, leptin is significantly released from cells. At 120 and 240 min, secretagogues enhance leptin secretion into the medium by 35% (P < 0.05) and 40% (P < 0.01), respectively. Cycloheximide prevents the synthesis and the secretagogue-mediated secretion of leptin. Monensin, an inhibitor of protein translocation, has no effect on leptin synthesis, but it blocks the secretagogue-mediated secretion of leptin. These findings suggest that secretagogues enhance leptin release by increasing leptin synthesis, rather than by enhancing the release of a preexisting cytosolic pool of leptin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS A 16-kDa protein that is secreted by adipocytes. It has pleiotropic functions, including the inhibition of appetite, the regulation of the neuroendocrine axes, the enhancement of thermogenesis, and the modulation of glucose sensitivity (for review, see Ref. 1). The regulation of secretion of this important homeostatic hormone has been intensively studied. Several circulating factors affect leptin gene expression and protein secretion (for review, see Refs. 1 and 2). In whole animal studies and in cultured cells, glucocorticoids, insulin, glucose, and cytokines increase leptin mRNA levels in adipocytes. ß-Adrenergic agonists, thiazolidinediones, and testosterone inhibit intracellular leptin mRNA levels.

The regulation of leptin secretion is incompletely understood. Ultrastructural studies have failed to show any large storage organelles for leptin within the adipocyte (3, 4). However, immunogold particles that represent leptin in the adipocytes in fat tissue were compartmentalized in alveolae that resemble vesicles (4). The functional significance of these histological findings is not known. We do not know, for instance, whether leptin secretagogues release hormone from cytosolic stores or enhance the constitutive secretion of leptin by increasing the synthesis, packaging, and trafficking through the cytosol. Several recent studies have provided compelling evidence for both insulin-mediated constitutive and regulated secretory pathways (3, 5, 6). Several studies (3, 7, 8) have demonstrated insulin-mediated increases in leptin gene transcription and synthesis; such studies highlight the importance of constitutive secretion. By contrast, evidence for regulated leptin secretion is based on two findings: 1) insulin stimulates leptin release from adipocytes within 10 min, a time frame too short for insulin-mediated leptin synthesis (3); and 2) insulin stimulates leptin release even in the presence of protein synthesis inhibitors (5, 6).

The purpose of the present study was to define the time course of synthesis and release of leptin from cultured adipocytes in the presence and absence of leptin secretagogues. Our results demonstrate that glucose, pyruvate, and insulin stimulate leptin release by increasing the intracellular synthesis of leptin. We did not observe a secretagogue-mediated quantal release of leptin from stored cytosolic pools.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of adipocytes and experimental protocol
All animals were humanely treated, and the experimental protocols were reviewed and accepted by the institutional animal care and use committee at Virginia Commonwealth University. Male Sprague Dawley rats (150–500 g; Harlan Sprague Dawley, Inc., Indianapolis, IN) were anesthetized with isoflurane before decapitation. We studied isolated adipocytes from the extensively studied epididymal fat pad (3, 9). This fat pad is easily accessible and is metabolically active; leptin gene expression from this site is modulated by fasting, refeeding, and insulin (8, 10). Leptin gene expression in epididymal fat is similar to expression in other fat stores (11, 12).

Epididymal fat pads were dissected away from the epididymis and other connective tissue. Epididymal fat pads are composed of a heterogeneous population of adipocytes. We found that the quantity of leptin released from adipocytes located near the tip of the fat pad is less than that released from the base of the fat pad (13). Therefore, we cut the epididymal fat pad into approximately two equal halves, and we discarded the distal half. The fat pad at the base was minced and incubated with 2.5 mg/ml collagenase; these procedures were similar to those described by Rodbell (14) and modified by Marshall (15). After the cells were filtered through a mesh and washed, they were concentrated by centrifugation, and the medium was removed. An aliquot of isolated adipocytes was withdrawn, and cell number was determined with a hemocytometer; the concentration of cells ranged from 1–4 x 106 cells/ml. Aliquots (230 µl) of cells were then added to equal volumes of medium (base DMEM, D5030, Sigma, St. Louis, MO) in 24-well culture dishes (16-mm wells) and incubated in a 37 C incubator (5% CO2). Base DMEM is identical to complete DMEM (D5523, Sigma), but contains no glucose, pyruvate, or L-glutamine. After 15 min in base medium, 4.6 µl of a 100x solution of pyruvate, glucose, and insulin were added to 460 µl cell suspension. The final concentrations of secretagogues in the cell suspension were as follows: pyruvate, 1 mM; glucose, 25 mM; and insulin (bovine, Sigma I-4011), 100 ng/ml. The measured glucose concentration in the medium was approximately 25% greater than the indicated glucose concentration in the cell suspension. The concentration of glucose in the medium changed minimally (<1%) during the longest incubation periods.

To measure the effects of inhibitors of protein synthesis and vesicle trafficking on leptin secretion, isolated adipocytes were incubated in base medium with vehicle (ethanol; final concentration, 0.1%), cycloheximide (final concentration, 25 µM), or monensin (final concentration, 50 µM). After 15 min, the cells were incubated in the absence or presence of leptin secretagogues as described above.

Measurement of intracellular leptin and leptin secretion
Zero, 15, 30, 60, 120, and 240 min after the addition of leptin secretagogues, cell suspensions were removed and placed in a microcentrifuge tube. The cells and medium were separated by centrifugation with a quick (<1 sec) flick of the on-off switch. Medium (230 µl) was withdrawn from each sample, and leptin levels were measured with the rat leptin RIA kit, as described by the supplier (Linco Research, Inc., St. Charles, MO). The cells in each sample were incubated with an equal volume (230 µl) of lysis buffer (1% Triton X-100, 0.3 M NaCl, 1 mM EDTA, and 0.05 M Tris, pH 7.4, with complete protease inhibitor cocktail, one tablet/10 ml; Roche Molecular Biochemicals, Indianapolis, IN). The cell suspension was vortexed intermittently for 15 min, then briefly centrifuged. The leptin concentration in the cell lysate was measured with the rat leptin RIA kit. The limits of sensitivity and linearity for the rat leptin assay are 0.5 ng/ml and 50 ng/ml, respectively. The interassay variation for the leptin assay at 1.6 ng/ml (Quality Control I) is 2.5%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The time courses of intracellular leptin accumulation and leptin secretion from isolated adipocytes into the medium in the absence (control) and presence of leptin secretagogues (glucose, pyruvate, and insulin) are shown in Table 1Go. In the absence of leptin secretagogues, total leptin in the culture system increased from 6.9 ± 0.2 ng/ml·106 cells at 0 min to 13.2 ± 0.8 ng/ml·106 cells at 240 min. These data suggest that even in the absence of GPI, isolated adipocytes actively synthesize leptin. In the presence of GPI, leptin synthesis increased markedly; total leptin in the system increased by 1.7-fold at 240 min.


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  		Table 1. Time course of synthesis, intracellular accumulation, and secretion of leptin

 
In control cells, the intracellular leptin concentration increased minimally from 5.2 ± 0.1 ng/ml·106 cells at 0 min to 6.4 ± 0.6 ng/ml·106 cells at 240 min. GPI increased the intracellular leptin concentration by approximately 2.0-fold, resulting in an intracellular leptin concentration of 13.2 ± 1.2 ng/ml·106 cells by 240 min. Significant GPI-mediated increases in intracellular leptin were observed at all time points; at 15, 30, 60, and 120 min, GPI increased intracellular leptin by 1.1-, 1.25-, 1.6-, and 1.8-fold respectively.

The leptin concentration in the medium rose steadily in control cells, from 1.7 ± 0.1 ng/ml·106 cells at 0 min to 6.8 ± 0.2 ng/ml·106 cells at 240 min (Table 1Go). In this experiment, GPI increased the leptin concentration in the medium to 9.5 ± 0.4 ng/ml·106 cells by 240 min; that is, leptin release increased by 1.4-fold. No difference was observed in the leptin concentration secreted from control cells or from cells incubated with GPI in the first 30 min. GPI significantly increased leptin secretion after 60 min of incubation.

To demonstrate the effect of GPI on the kinetics of the synthesis, intracellular accumulation, and secretion of leptin, we plotted the differences in leptin concentrations from GPI-treated and control cells, as shown in Fig. 1Go. GPI rapidly increased leptin synthesis, as demonstrated by an immediate rise in the total leptin concentration in the cell suspension. In the first 30 min, the rates of GPI-mediated leptin synthesis (total) and intracellular accumulation were identical as newly synthesized leptin accumulates within the cell. After 30 min, GPI stimulated leptin secretion, as shown by an appearance of leptin in the medium by 60 min. After 60 min, the rate of GPI-mediated intracellular accumulation of leptin slowed, whereas GPI progressively stimulated leptin secretion into the medium.



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  		Figure 1. Effects of GPI on synthesis, intracellular accumulation and secretion of leptin. The experiment was performed as described in Table 1Go. Each data point represents the mean ± SEM (n = 3) of the difference in leptin concentrations in cells incubated with and without glucose, pyruvate, and insulin. *, P < 0.05, GPI-treated cells vs. control cells; **, P < 0.01.

 
To determine whether secretagogue-induced leptin secretion occurs independently from leptin synthesis, we measured the secretion, intracellular accumulation, and synthesis of leptin in the absence and presence of the protein synthesis inhibitor, cycloheximide. As shown in Fig. 2Go, we again demonstrate that GPI stimulated leptin synthesis (Fig. 2AGo), intracellular accumulation (Fig. 2BGo), and secretion from isolated adipocytes (Fig. 2CGo). Cycloheximide effectively turned off leptin synthesis (Fig. 2AGo) in the absence and presence of GPI. In the absence of leptin synthesis, intracellular leptin gradually declined (Fig. 2BGo), and leptin was slowly released into the medium (Fig. 2CGo). Cycloheximide prevented GPI-mediated leptin release into the medium (Fig. 2CGo).



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  		Figure 2. Effect of cycloheximide on the kinetics of leptin secretion into the medium and intracellular accumulation. Adipocytes were incubated with vehicle (VH; closed symbols) or cycloheximide 25 µM (CHX; open symbols), in the absence (squares) or presence (triangles) of glucose, pyruvate, and insulin (GPI) as described in Materials and Methods. After various times, the intracellular (middle panel) and medium (right panel) leptin concentrations were determined. The total (left panel) leptin concentration represents addition of the leptin concentration in the medium plus the intracellular leptin concentration. Each data point represents the mean leptin concentration (nanograms per ml per 106 cells) ± SEM from three separate experiments. *, P < 0.05, by unpaired t test, GPI-treated cells vs. control cells; **, P < 0.01.

 
We next incubated adipocytes with monensin, a drug that inhibits vesicular trafficking. The effects of monensin on the time course of total synthesis, intracellular accumulation, and secretion of leptin in the absence and presence of GPI are shown in Fig. 3Go. Unlike control cells, cells incubated with monensin showed evidence of cytolysis at 240 min. Therefore, data at 240 min were eliminated from analysis. Monensin had little effect on total leptin synthesis; the total leptin concentrations in control cell suspensions incubated with monensin were almost identical to the total leptin concentrations in cell suspensions not incubated with monensin (Fig. 3Go, left panel). Furthermore, monensin had no effect on GPI-mediated leptin synthesis (Fig. 3Go, left panel). Monensin effectively inhibited the release of leptin from cells incubated without and with GPI (Fig. 3Go, right panel). As monensin inhibited secretion, but had no effect on synthesis, the predicted effect on intracellular leptin was observed; monensin caused a marked increase in the intracellular content of leptin in the presence and absence of GPI (Fig. 3Go, middle panel).



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  		Figure 3. Effect of monensin on the kinetics of leptin secretion and intracellular leptin accumulation. The experiment was performed exactly as described in Fig. 1Go, except cells were also incubated without and with monensin (50 µM) as described in Materials and Methods. Each data point represents the mean leptin concentration (nanograms per ml/106 cells) ± SEM from three separate experiments. *, P < 0.05, by unpaired t test, GPI-treated cells vs. control cells; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We studied the effects of leptin secretagogues on the kinetics of leptin secretion in cultured rat adipocytes. We found that under basal conditions, leptin is slowly, but continuously, synthesized and secreted; the rate of synthesis approximately equals the rate of secretion, and little or no leptin accumulates in the cells. Pyruvate, glucose, and insulin rapidly stimulate leptin synthesis. Within the first 30 min, these secretagogues increase total leptin production without stimulating leptin secretion concomitantly, and hence, the intracellular hormone level increases. By 60 min, the high rate of leptin synthesis continues, but adipocytes begin to secrete leptin, and the rate of intracellular accumulation of leptin slows. By 120 min, a new steady state is reached, during which the rate of leptin synthesis equals the rate of leptin secretion, and intracellular leptin accumulation plateaus. Therefore, the increase in leptin secretion induced by pyruvate, glucose, and insulin appears to be mediated principally by the increase in leptin synthesis.

We did not observe measurable secretagogue-mediated leptin secretion, either as an acute fall in intracellular leptin concentration or as an increase in the leptin concentration in the medium, within the first 30 min. Our data, therefore, do not support secretagogue-mediated release of a stored intracytosolic pool of leptin. The 30- to 60-min interval required for secretagogue-mediated leptin release may be consistent with the time necessary for the leptin gene to be transcribed and translated and for leptin to be packaged and transferred from the synthetic machinery to the cell surface and released. Other theories may explain the observed kinetic data. For instance, perhaps leptin secretagogues cleave an immunoinactive proleptin form of the hormone that is stored intracellularly. The increase in intracellular immunoactive leptin would be observed when adipocytes are incubated with secretagogues. We find this theory less tenable than the theory that secretagogues are inducing leptin synthesis for the following reasons. Firstly, to our knowledge, a proleptin hormone has not been identified to date. Secondly, one would expect to observe a concomitant rise in constitutive release of leptin as prohormone cleavage makes more leptin available for secretion. Instead, we find that secretagogue-induced intracellular leptin accumulates intracellularly before release into the medium. The concentrations of glucose and insulin in this study were high and were chosen to maximize leptin secretion. We hypothesize that lower concentrations of glucose would stimulate leptin secretion by similar mechanisms because previous work in our laboratory has demonstrated that glucose mediates leptin gene expression and secretion in a dose-responsive manner (13). However, measurements of total, intracellular, and medium leptin concentrations with lower concentrations of glucose would need to be performed to conclude this with certainty.

Our findings are consistent with other reported studies of insulin-mediated leptin secretion in isolated rat adipocytes. Several laboratories have reported that insulin stimulates leptin gene expression (8, 13, 16, 17). Hardie et al. (18) found that insulin-stimulated leptin release by mature adipocytes can be detected within 1 h of culture. In their studies the transcription inhibitor, actinomycin D, inhibited insulin- mediated leptin release; this finding suggests that insulin- induced leptin secretion required de novo transcription. Actinomycin D inhibition of leptin release has not been found consistently (5, 19). Gettys et al. (20) and Cheng et al. (21) demonstrated insulin-mediated leptin secretion within 30 min, a time frame still consistent with de novo synthesis and intracellular trafficking before secretion.

In contrast to the above findings, Barr et al. (3) demonstrated that insulin could induce leptin secretion within 10 min, a time frame that is too short for insulin induction of transcription, translation, trafficking, and secretion of leptin, but that is consistent with the release of intracellular stores of leptin. In the study by Barr et al., intracellular leptin was measured by immunofluorescence. They found that after 10 min of incubation, the amount of leptin remaining in insulin-treated tissue was less than the amount in untreated tissue. Concomitant with the fall in the intracellular leptin concentration, its concentration in the medium after 10 min of insulin treatment was significantly greater than that in untreated adipose tissue. In the present study we did not detect a secretagogue-mediated decline in intracellular leptin concentration or a rise in secreted leptin within 10 min. The immunofluorescence technique may be more sensitive in detecting a small change in intracellular leptin concentration than is the RIA technique we used in the present study. However, the difference is our observations is probably explained by the different culture systems. In our culture system we did not observe a fall in intracellular leptin concentration. Rather, intracellular leptin concentrations were stable in the basal state, and they increased in response to leptin secretagogues.

We have shown that the protein synthesis inhibitor, cycloheximide, completely blocks secretagogue-mediated leptin secretion. This finding along with our supporting kinetic data provide strong evidence that pyruvate, glucose, and insulin increase leptin secretion by enhancing leptin synthesis. We were unable to detect a secretagogue-mediated release of leptin from a cytosolic pool. Two other laboratories, however, reported that insulin (5) or serum (6) induces leptin secretion in the presence of cycloheximide. Hence, the researchers concluded that leptin is secreted from a preexisting intracellular pool. The major difference in methodology between the present study and the two previous studies is that the isolated adipocytes were incubated with cycloheximide for only 20 min in the two previous studies. The cells were then washed and reincubated for 1–2 h in the presence or absence of insulin or serum. Neither of the previous groups included controls that showed effective inhibition of protein synthesis; therefore, in the reincubation medium, insulin or serum may have stimulated protein synthesis. In our study cells were incubated continuously with cycloheximide, and we found that leptin synthesis was fully inhibited.

Several laboratories have characterized the subcellular localization of leptin in adipocytes. Barr et al. (3) showed that leptin immunostaining displayed a honeycomb pattern, typical of proteins in the endoplasmic reticulum of adipose cells. Leptin was never seen in a punctate staining pattern indicative of storage in secretory vesicles. Bornstein et al. (4) detected immunogold particles that represented leptin along cell membranes and small clustering in alveolate structures of uniform size (40–80 nm in diameter) attached to the cell membrane. No large storage organelles for leptin were detected in the adipocyte. Treatment of adipocytes with Brefeldin A blocked leptin secretion into the medium, which led to massive intracellular accumulation (17). Brefeldin A inhibits the translocation of secretory proteins from the endoplasmic reticulum to the Golgi apparatus. We found that monensin also blocked leptin secretion and resulted in the intracellular accumulation of the hormone. Monensin had no effect on protein synthesis. Monensin is an ionophore that induces the movement of sodium into cellular compartments in the central vacuolar system by sodium/hydrogen ion exchange. This process increases the pH, and thus impairs protein sorting and transport in the trans-Golgi complex and inhibits vesicle formation (22).

From these ultrastructural and biochemical findings we speculate that leptin transverses a classical secretory pathway within adipocytes. After synthesis, the secretory protein is packaged within the Golgi apparatus into small alveolate vesicles. Under basal conditions, the vesicles release leptin into the surrounding tissue as fast as leptin is synthesized. In the presence of pyruvate, glucose, and insulin, adipocytes synthesize more leptin, and the Golgi apparatus packages more product into secretory vesicles. Intracellular leptin accumulates until a new steady state is reached, during which the quantity of secreted leptin equals the quantity of synthesized leptin.


   Acknowledgments
 
Received January 16, 2001. Accepted April 16, 2001.

Address all correspondence and requests for reprints to: Dr. James R. Levy, McGuire Veterans Administration Medical Center 111-P, 1201 Broad Rock Boulevard, Richmond, Virginia 23249. E-mail: james.levy@ med.va.gov.

This work was supported by the V.A. Merit Review Board (to J.R.L.).


   Footnotes
 
Abbreviations: GPI, Glucose, pyrvate, insulin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  10. Mizuno TM, Bergen H, Funabashi T, Kleopoulos SP, Zhong Y-G, Bauman WA, Mobbs CV 1996 Obese gene expression: reduction by fasting and stimulation by insulin and glucose in lean mice, and persistent elevation in acquired (diet-induced) and genetic (yellow agouti) obesity. Proc Natl Acad Sci USA 93:3434–3438[Abstract/Free Full Text]
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  19. Fain JN, Bahouth SW 1998 Stimulation of leptin release by actinomycin D in rat adipocytes. Biochem Pharmacol 55:1309–1314[CrossRef][Medline]
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