The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, 45Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased 45Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.
- calcium entry
- intracellular calcium
leptin is an adipocytokine produced mainly by white adipocytes, and to a lesser extent by other tissues such as the placenta (1, 19, 43). Its concentration in the plasma is tightly correlated with the total amount of white adipose tissue in the body (11). One of its main sites of action is located within the hypothalamus, where it acts to regulate energy expenditure, food intake, and the activity of the sympathetic nervous system, at least in rodents. Leptin has therefore been considered as a “lipostatic factor” contributing to the regulation of body weight via a negative feedback loop (17).
Many polypeptidic hormones, such as insulin, glucagon, ACTH, or thyrotropin-stimulating hormone, are secreted from endocrine tissues in response to extracellular stimuli via a mechanism that involves calcium as a necessary second intracellular messenger (14, 23). Increases in intracellular calcium concentrations are achieved either by releasing calcium from intracellular stores, such as endoplasmic reticulum, or by triggering calcium entry from the extracellular medium through specific channels (34). White adipose tissue shares some characteristics of endocrine tissues, such as preformed vesicles containing leptin in the cytosol and functional L-type calcium channels on the plasma membrane (3, 13). Flask-shaped vesicles, 60–80 nm in diameter, localized close to the plasma membrane have been revealed by electron microscopy and immunochemistry (5, 8). It is also known that insulin is a potent stimulator of leptin secretion in vitro and in vivo (6, 15, 22) and that insulin action can be inhibited by norepinephrine (7) or long-chain fatty acids (6). Although extensive studies have been carried out on the role of calcium on the secretion of many polypeptic hormones (23), calcium regulatory effects on leptin secretion are still not well understood. Only one study reported that the presence of extracellular calcium is necessary to observe a stimulation of leptin secretion by insulin (24). However, the detailed mechanism by which calcium regulates leptin secretion appears to not have been studied. In particular, it is not known whether calcium modulates leptin secretion by affecting glucose metabolism, insulin signaling, leptin synthesis, and/or exocytosis per se.
To investigate the influence of calcium on insulin-stimulated leptin secretion, white adipocytes were incubated in various media (with or without calcium) containing different energetic substrates (glucose or pyruvate) in the presence of insulin and/or agents modulating membrane calcium permeability. Extracellular leptin was measured by RIA, and calcium uptake was evaluated using 45Ca. We are now the first to report that calcium has a permissive and indirect role on insulin-stimulated leptin secretion by regulating the early steps of glucose metabolism. Insulin does not trigger intracellular calcium uptake. On the contrary, the present data suggest that increasing cytosolic calcium entry from intracellular or extracellular stores has a potent inhibitory effect on insulin-stimulated leptin secretion. Thus the role of calcium in the mechanism of stimulus-secretion coupling of leptin release in adipocytes is markedly different from that seen in the secretion of most peptidic hormones from other systems.
MATERIALS AND METHODS
Fatty acid-free BSA, collagenase (type II, lot 107H8649), nitrendipine, nimodipine, nifedipine, A-23187, pyruvate, glucose, CaCl2, KCl, EGTA, W-13, cycloheximide, cytochalasin B, phloretin, Triton X-100, phenylmethanesulfonyl fluoride (PMSF), aprotinin, leupeptin, pepstatin, ATP, and thapsigargin were all obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). Insulin (Humulin-R) was purchased from Eli Lilly (Toronto, Ontario, Canada). 45Ca and Ecolite were bought from ICN Biochemicals (Costa Mesa, CA). Dinonyl phthalate was acquired from VWR (TCI, Portland, OR).
Male Wistar rats were obtained from Charles River (St. Constant, Quebec, Canada) and were housed in individual cages at 24°C with a 12:12-h light-dark cycle. The rats received standard Purina chow and water ad libitum. The mean body mass of the rats used in the present experiments was 300 ± 15 g.
Adipocytes were isolated from epididymal fat pads by a slight modification of Rodbell's (32) method. Briefly, rats were killed by decapitation, and their epididymal fat pads were removed and placed in Krebs-Ringer bicarbonate buffer (KRB) of the following composition: 120 mM NaCl, 4.75 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 5.5 mM glucose, 20 mM HEPES, and 1% fatty acid-free BSA (1% KRB), pH 7.4, with or without CaCl2 (2.5 mM). The minced tissue was incubated in fresh medium containing 0.5 mg/ml collagenase at 37°C for 15–20 min with a shaking frequency of 150 cycles/min. At the end of the incubation, the cells were filtered through a 500-μm nylon filter (Nitex) and diluted with 5 ml 1% KRB. The floating cells were washed four times with the same medium and preincubated at 37°C for 15 min (shaking frequency of 40 cycles/min). Next, they were washed three times with warm (37°C) KRB containing 4% fatty acid-free BSA (4% KRB). For studies performed in the absence of calcium, cells were washed three times with a calcium-free medium. Adipocytes were incubated under the same conditions for 2 h (unless otherwise specified) in the presence of agents to be tested, with a cell concentration of 3–5 × 105 cells/4% KRB. The adipocytes were then allowed to float, and the infranatants were frozen at −20°C for leptin and glycerol measurements.
This method was adapted from Martin et al. (28). After isolation, one-half of the cells was used for stimulation of leptin secretion and one-half for the measurement of calcium uptake. Cells were kept in a plastic vial in 4% KRB with calcium (2.5 mM), at 37°C. At time 0, agents to be tested, including 1 μCi 45Ca (1 mCi/μmol), were added to the vials and incubated at 37°C under gentle agitation. At chosen times (see Fig. 5), 200-μl samples of cells (3–5 × 105 cells/ml) were taken and deposited in a long narrow plastic tube containing 100 μl dinonyl phthalate. The tubes were centrifuged at 12,000 g for 1 min and then cut in two at the level of the dinonyl phthalate layer. The upper layer containing the cells and a volume of 100 μl of the lower layer were put in two vials with 10 ml counting liquid (Ecolite; ICN). After 1 h of shaking, radioactivity was counted with an LKB scintillation counter (1215 Rackbeta). Uptake values were corrected for the 45Ca trapped in the extracellular water space using [U-14C]sucrose as a control. 45Ca uptake results were expressed as a percentage of total cell radioactivity over the total medium radioactivity.
Measure of intracellular leptin content.
Adipocytes were shaken for 1 min (vortex) in the presence of 1% Triton X-100 and a mixture of the following protease inhibitors: 200 μM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 5 μg/ml aprotinin. After overnight incubation at 4°C, lysates were centrifuged for 5 min at 12,000 g, and the infranatants were assessed for leptin concentration.
Leptin and glycerol assays.
Leptin concentrations were determined by RIA using a kit available from Linco Research (St. Charles, MO). Glycerol was measured using an enzymatic method (37).
Effects of a calcium-free medium on basal and insulin-stimulated leptin secretion.
To assess the role of calcium on leptin secretion, isolated white adipocytes were incubated for 2 h in KRB (4% albumin) containing either calcium (2.5 mM) or no calcium and 100 μM of the specific calcium chelator EGTA. Insulin (10 nM) was added in both media to stimulate maximally leptin secretion. At the end of the incubation, infranatants were removed and analyzed for leptin and glycerol concentrations. In addition, cellular leptin content was measured as described in materials and methods. In the presence of calcium, insulin stimulated leptin secretion (Fig. 1A) and increased intracellular leptin content in white adipocytes (Fig. 1B). In contrast, the lack of calcium completely prevented the insulin-stimulating effects on intra- and extracellular leptin concentrations. However, in the absence of insulin, the lack of calcium did not affect secretion, suggesting that calcium specifically affects stimulated exocytosis rather than basal secretion. It should be noted that calcium is known to have antilipolytic effects by activating phosphodiesterases (42). Therefore, the absence of calcium may have elicited an increase in basal lipolysis, leading to an inhibition of insulin-stimulated leptin secretion (6, 15). Nevertheless, measurements of extracellular glycerol in the experiments described in Fig. 1 (results not shown) revealed that lipolysis was not modified in a calcium-free medium.
Effect of pyruvate on leptin secretion in calcium-free conditions.
The presence of glucose and calcium in the extracellular medium has been shown to be essential for observing the insulin stimulatory effects on leptin secretion (24, 29). It is also known that fusion of GLUT4-containing vesicles with the plasma membrane is highly dependent on calcium and calmodulin activation by calcium (36). However, the detailed mechanisms by which glucose and calcium regulate leptin exocytosis are still not well understood. A lack of extracellular calcium might prevent insulin-stimulated leptin secretion by decreasing the availability of glucose as an energy source for leptin synthesis and/or by directly inhibiting leptin exocytosis. To test these hypotheses, insulin-stimulated glucose uptake was bypassed using pyruvate, a glycolytic intermediate that enters the cell independently of transporters. Leptin secretion in the presence of glucose or pyruvate was similar (Fig. 2), suggesting that pyruvate can efficiently replace glucose as an energy source. However, in a calcium-free medium, insulin enhanced leptin secretion only when pyruvate, but not glucose, served as an energy source (Fig. 2B). This suggests that calcium acts at metabolic steps preceding pyruvate oxidation. It should be pointed out that pyruvate did not stimulate basal leptin secretion, nor did it potentiate insulin stimulation in the presence of glucose (Fig. 2). To test whether calcium acted at the level of glucose transport, we used the following three inhibitors of intracellular glucose entry: cytochalasin B inhibits facilitative glucose uptake at the level of GLUT1 and -4 (21); phloretin specifically inhibits Na-independent d-glucose transporters of the GLUT family (35); and W-13 is a calmodulin antagonist, inhibiting glucose transport in 3T3-L1 cells (18, 36). All three inhibitors suppressed insulin-stimulated leptin secretion in the presence of glucose (Fig. 3). As expected, all agents failed to inhibit insulin-stimulated leptin secretion when pyruvate was used as an energy source. These results suggest that the lack of calcium affects insulin-stimulated leptin secretion by inhibiting mobilization of the glucose transporters rather than by affecting distal steps of leptin exocytosis.
Effect of cycloheximide on insulin-stimulated leptin secretion.
The stimulatory effects of insulin on leptin secretion and synthesis are both abolished by the lack of extracellular calcium when glucose is used as a substrate (Fig. 1). To determine whether de novo leptin synthesis is required to observe the stimulatory action of insulin on leptin secretion, cells were incubated in the presence of cycloheximide, a protein synthesis inhibitor. These experiments were carried out in the presence and absence of calcium using glucose or pyruvate as an energy source (Figs. 4, A and B). Under all conditions, cycloheximide completely prevented the stimulatory action of insulin on both leptin synthesis and secretion. Cycloheximide also inhibited basal leptin secretion (data not shown), in agreement with previous reports showing that leptin is continuously synthesized and secreted (25). These results, combined with the data described in Fig. 1, show that de novo leptin synthesis is required to observe a stimulation of leptin secretion by insulin. It might be pointed out that cycloheximide, in addition to inhibiting protein synthesis, may inhibit lipolysis by activating α1-adrenoceptors (38). However, we previously reported that antilipolytic agents do not affect basal leptin secretion in adipocytes (6). It is therefore likely that cycloheximide acted mainly by directly inhibiting protein synthesis. The observation that, in the absence of calcium, insulin enhances leptin synthesis when pyruvate (as opposed to glucose) is used as a substrate suggests that insulin does not stimulate leptin synthesis in a calcium-dependent manner. This is in accordance with our previous results (see Figs. 2 and 3) showing that calcium specifically affects leptin release at an early step of glucose metabolism.
Measurements of calcium entry in the presence or absence of insulin.
An increased calcium uptake is a necessary step for the secretion of many polypeptidic hormones. To assess whether insulin stimulates calcium uptake in adipocytes, cells were incubated in 4% KRB containing 2.5 mM calcium, 1 μCi 45Ca, 5 mM glucose, and 0 or 10 nM insulin. Samples were taken at various times for analysis (see materials and methods). In spite of the fact that insulin stimulated leptin secretion on the same experiments (results not shown), insulin did not alter calcium uptake at all time points (Fig. 5). On the other hand, specific L-type calcium channel inhibitors, such as nifedipine, nitrendipine, or nimodipine (100 nM-1 mM), did not inhibit insulin-stimulated leptin secretion (results not shown). These observations indicate that stimulated calcium uptake is not required for the enhanced leptin secretion.
Effect of an increase in calcium uptake on leptin secretion.
To further assess the role of calcium on leptin secretion, cells were incubated with several agents known to increase calcium uptake by acting on calcium permeability of the plasma membrane. Insulin (10 nM)-stimulated leptin secretion was inhibited by incubating the cells in the presence of high concentrations of calcium (10–30 mM), with the calcium ionophore A-23187 (100 μM; see Ref. 31), or by activating calcium-ATPase pumps with ATP (1.6 mM; see Ref. 4 and Fig. 6A). Leptin synthesis was also inhibited (results not shown). Calcium uptake measurements in the same experiments revealed that calcium, A-23187, and ATP increased calcium uptake (Fig. 6B). Nifedipine (100 μM), as well as nitrendipine and nimodipine (results not shown), did not alter leptin secretion or calcium uptake. Thus, in white adipocytes, calcium entry inhibits leptin secretion, whereas in other systems (Langerhans islets and others) it usually stimulates hormonal secretion.
Effect of intracellular calcium store release on leptin secretion.
Releasing endogenous calcium from mitochondria and the endoplasmic reticulum can raise intracellular calcium levels. To examine if the release of intracellular calcium stores can inhibit leptin secretion, white adipocytes were incubated in the presence of increasing concentrations of thapsigargin. This agent inhibits intracellular Ca2+-ATPase, thus increasing intracellular calcium by preventing the reuptake of cytosolic calcium (26). Thapsigargin dose-dependently inhibited insulin-stimulated leptin secretion with no effect on basal secretion (Fig. 7). These results suggest that a sustained release of endogenous calcium can also inhibit insulin-stimulated leptin secretion.
Effect of pyruvate on agents increasing calcium uptake.
An increase in intracellular calcium levels decreases insulin-stimulated glucose uptake and interferes with insulin signaling (4). To determine whether an excess of calcium inhibits insulin-stimulated leptin secretion by interfering with the early steps of glucose metabolism, cells were incubated in a medium containing 5 mM glucose, 2.5 mM calcium, 10 nM insulin, and one of the following agents: calcium (final concentration 10 or 30 mM), ATP (1.6 mM), A-23187 (200 μM), or thapsigargin (10 μM). Pyruvate (5 mM) was added to bypass the early steps of glucose metabolism (see Figs. 2 and 3). However, the addition of pyruvate was unable to restore insulin-stimulated leptin secretion (Fig. 8). Thus high levels of intracellular calcium may inhibit insulin-stimulated leptin secretion at metabolic steps that are independent of glucose metabolism.
The principal goal of this study was to investigate the mechanisms by which calcium regulates insulin-stimulated leptin secretion. Previous observations were rather limited and inconclusive: one study reported that the presence of calcium in the extracellular medium is required for leptin exocytosis (24), whereas another reported that calcium ionophores do not affect leptin secretion (33). The observation that calcium is required for insulin-stimulated secretion, but not for basal secretion (Fig. 1), indicates that calcium does not exert a generalized effect on leptin release and that it affects secretion by specifically acting on metabolic pathways controlled by insulin. In particular, insulin increases glucose uptake by stimulating fusion of preformed vesicles containing GLUT4 (a specific glucose tansporter) to the plasma membrane. This event is highly dependent on the presence of calcium (10, 36). On the other hand, glucose uptake has been shown to be essential for insulin-stimulated leptin secretion (25, 29). We therefore hypothesized that calcium modulates leptin secretion by acting on some early steps of glucose metabolism, most probably at the level of glucose uptake. This hypothesis is supported by the following observations. First, insulin did not stimulate leptin secretion or synthesis when cells were incubated in a calcium-free medium containing glucose (Fig. 1). Second, insulin stimulated leptin secretion in the presence of pyruvate and in the absence of glucose, independently of the presence of calcium in the medium. Because pyruvate permeates the plasma membrane without the use of a specific transporter, the latter observation suggests that calcium affects secretion by acting on a metabolic step upstream of pyruvate oxidation. It may be pointed out that pyruvate, per se, did not trigger leptin release (Fig. 2). Third, cytochalasin B, phloretin, and W-13, three inhibitors of glucose uptake, inhibited insulin-stimulated leptin secretion in the presence of glucose but were ineffective in the presence of pyruvate (Fig. 3). Fourth, de novo leptin synthesis is required to observe the stimulatory effect of insulin on leptin secretion independent of the presence of calcium in the medium (Fig. 4). On the whole, these observations suggest that calcium is necessary for insulin-stimulated leptin secretion by affecting glucose uptake.
Although the presence of calcium appears to be required for insulin-stimulated leptin secretion, there are important differences between the effect of calcium on leptin secretion and the classical exocytotic release of many polypeptidic hormones, such as insulin, glucagon, somatostatin, etc. For instance, insulin did not stimulate 45Ca uptake by adipocytes (Fig. 5), whereas it is well established that insulin secretagogues acutely stimulate calcium uptake in pancreatic secretory cells (2). Furthermore, blockers of L-type calcium channels that prevent calcium entry into adipocytes (nitrendipine, nimodipine, and nifedipine; see Refs. 10, 13, and 27) did not affect insulin-stimulated leptin secretion (results not shown). On the contrary, we found that an increase in calcium uptake induced by A-23187, ATP, or high concentrations of calcium inhibit insulin-stimulated leptin secretion (Fig. 6), whereas, in β-cells, calcium ionophores or ATP causes a sustained release of insulin (30, 38). Likewise, thapsigargin, an agent that increases cytosolic calcium, inhibits stimulated leptin secretion (Fig. 7), whereas it potentiates the stimulatory effects of glucose on insulin release from pancreatic islets (41). Finally, pyruvate (in the absence of glucose) did not reverse the inhibitory effects of A-23187, ATP, or high concentrations of extracellular calcium (Fig. 8), suggesting that an elevated intracellular calcium concentration might affect leptin secretion at metabolic steps distal to substrate oxidation. Nevertheless, it is still possible that calcium may also downregulate insulin signaling (9, 20), since it has been reported on 3T3-L1 preadipocyte cultures in short-term incubations (40). Thus there are many metabolic differences between the effects of calcium on leptin secretion from adipocytes and classical exocytosis.
Morphological studies also suggest that leptin is secreted via a distinct mechanism. Leptin is not stored in large secretory granules like many other polypeptidic hormones that are rapidly released. Immunohistological studies revealed that leptin is mainly stored in the endoplasmic reticulum and travels in small vesicles to the plasma membrane (3, 5, 8). Moreover, the pool of cytosolic leptin is small and not sufficient to account for the secreted amount observed. Another major difference between the mechanisms regulating insulin and leptin secretion is that leptin secretion is driven by de novo synthesis (Fig. 4), whereas insulin secretion is insensitive to protein synthesis inhibitors such as cycloheximide (16, 25). We did not observe insulin-induced alterations in leptin mRNA levels, at least in the short term (7).
Little is known about the regulatory function of calcium in relation with leptin secretion in humans, although some laboratories investigated the role of cytosolic calcium in insulin resistance (9, 10). Insulin-resistant obese individuals have elevated plasma leptin levels, and it might be argued that calcium insensitivity contributes to increase leptin secretion. However, the elevated leptin concentration in insulin-resistant obese individuals is mainly caused by an increased basal secretion resulting from adipocyte hyperthrophy and/or hyperplasia (11, 17) . In the present study, we found that calcium does not affect basal secretion, at least under short-term conditions. Nevertheless, it is likely that calcium plays an important regulatory role in humans, for instance during starvation-refeeding that acutely affects leptin secretion (15), but this remains to be investigated.
In summary, the present study provides evidence, for the first time, that calcium is essential for the stimulatory effect of insulin on leptin secretion through its permissive role on glucose uptake. Calcium does not affect leptin synthesis or exocytosis per se. Excess calcium disrupts leptin secretion by interfering with metabolic events that are independent from glucose uptake. Stimulation of leptin secretion by insulin does not require enhanced calcium uptake from the extracellular medium, nor calcium release from intracellular stores. This is in marked contrast to the mechanisms regulating the secretion of many polypeptidic hormones where extra- and intracellular calcium exerts a positive role on exocytosis.
This work was supported by grants from the Canadian Institutes of Health Research and the Association du Diabète du Québec.
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