Insulin-stimulated Leptin Secretion Research Articles

Insulin-stimulated leptin secretion requires calcium and PI3K/Akt activation

Numerous studies have focused on the regulation of leptin signalling and the functions of leptin in energy homoeostasis; however, little is known about how leptin secretion is regulated. In the present study we studied leptin storage and secretion regulation in 3T3-L1 and primary adipocytes. Leptin is stored in membrane-bound vesicles that are localized predominantly in the ER (endoplasmic reticulum) and close to the plasma membrane of both 3T3-L1 and primary adipocytes. Insulin increases leptin secretion as early as 15min without affecting the leptin mRNA level. Interestingly, treatment with the protein synthesis inhibitor cycloheximide and the ER-Golgi trafficking blocker Brefeldin A inhibit both basal and ISLS (insulin-stimulated leptin secretion), suggesting that insulin stimulates leptin secretion by up-regulating leptin synthesis and that leptin-containing vesicles go through the ER-Golgi route. The PI3K (phosphoinositide 3-kinase)/Akt, but not MAPK (mitogen-activated protein kinase), pathway is involved in ISLS invitro and invivo. Although Ca2+ triggers synaptic vesicle and secretory granule exocytosis, Ca2+ influx alone is not sufficient to induce leptin secretion. Remarkably, Ca2+ is required for ISLS possibly due to its involvement in insulin-stimulated Akt phosphorylation. We conclude that insulin stimulates leptin release through the PI3K/Akt pathway and that Ca2+ is required for robust Akt phosphorylation and leptin secretion.

The novel endocrine disruptor tolylfluanid impairs insulin signaling in primary rodent and human adipocytes through a reduction in insulin receptor substrate-1 levels

Emerging data suggest that environmental endocrine disrupting chemicals may contribute to the pathophysiology of obesity and diabetes. In a prior work, the phenylsulfamide fungicide tolylfluanid (TF) was shown to augment adipocyte differentiation, yet its effects on mature adipocyte metabolism remain unknown. Because of the central role of adipose tissue in global energy regulation, the present study tested the hypothesis that TF modulates insulin action in primary rodent and human adipocytes. Alterations in insulin signaling in primary mammalian adipocytes were determined by the phosphorylation of Akt, a critical insulin signaling intermediate. Treatment of primary murine adipose tissue in vitro with 100nM TF for 48h markedly attenuated acute insulin-stimulated Akt phosphorylation in a strain- and species-independent fashion. Perigonadal, perirenal, and mesenteric fat were all sensitive to TF-induced insulin resistance. A similar TF-induced reduction in insulin-stimulated Akt phosphorylation was observed in primary human subcutaneous adipose tissue. TF treatment led to a potent and specific reduction in insulin receptor substrate-1 (IRS-1) mRNA and protein levels, a key upstream mediator of insulin's diverse metabolic effects. In contrast, insulin receptor-β, phosphatidylinositol 3-kinase, and Akt expression were unchanged, indicating a specific abrogation of insulin signaling. Additionally, TF-treated adipocytes exhibited altered endocrine function with a reduction in both basal and insulin-stimulated leptin secretion. These studies demonstrate that TF induces cellular insulin resistance in primary murine and human adipocytes through a reduction of IRS-1 expression and protein stability, raising concern about the potential for this fungicide to disrupt metabolism and thereby contribute to the pathogenesis of diabetes.

Increased leptin storage with altered leptin secretion from adipocytes of rats with sucrose-induced dyslipidemia and insulin resistance: effect of dietary fish oil

This study examined the effect of long-term feeding a high-sucrose diet (SRD) on the modulation of rat adipocyte's leptin secretion and storage. For this purpose, we analyzed ( a) basal and insulin-stimulated leptin release and the role of isoproterenol and palmitate on insulin-stimulated leptin secretion, ( b) the correlation between leptin and glycerol released, ( c) the relationship between leptin contents and adiposity, and ( d) the effect of fish oil (FO) administration on the above parameters. Wistar rats were fed an SRD for 6 months. Whereas half the animals continued with SRD up to month 8, the other half was fed an SRD in which FO partially replaced corn oil from months 6 to 8. Total leptin release was reduced both basally and under insulin stimulation in SRD-fed rats. However, the ratio of leptin released after hormone stimulation to basal leptin levels was similar in the 3 dietary groups. Isoproterenol inhibited insulin-stimulated leptin release in the 3 groups, but the percentage was lower in the SRD. Palmitic acid mimicked the effect of isoproterenol. Leptin release from adipocyte of SRD-fed rats negatively correlated with glycerol release. Leptin store increased in fat pads of SRD and positively correlated with adiposity. Fish oil reduced leptin content and fat pad hypertrophy, and normalized basal lipolysis, leptinemia, and glucose homeostasis. This suggests that enhanced lipolysis and altered insulin sensitivity could play a role in the decrease of leptin released in SRD-fed rats. This is consistent with the reversion of all the alterations after FO administration.

Leptin and cardiovascular disease: response to therapeutic interventions.

Leptin and cardiovascular disease: response to therapeutic interventions.

Effect of altitude on leptin levels, does it go up or down?

reports of a relationship between altitude and circulating levels of leptin suggest a possible role of hypoxia in leptin regulation. However, studies on the changes in plasma leptin levels at altitude are controversial, with some showing an increase and others suggesting no change or a fall in these

Linoleic Acid Decreases Leptin and Adiponectin Secretion from Primary Rat Adipocytes in the Presence of Insulin

Obesity rates have dramatically increased over the last few decades and, at the same time, major changes in the type of fatty acid intake have occurred. Linoleic acid, an n-6 polyunsaturated fatty acid, is an essential fatty acid occurring in high amounts in several western diets. A potential role of this fatty acid on obesity has been suggested. Controversial effects of linoleic acid on insulin sensitivity have also been reported. Thus, the aim of this study was to examine the direct effects of linoleic acid on leptin and adiponectin production, two adipokines known to influence weight gain and insulin sensitivity. Because insulin-stimulated glucose metabolism is an important regulator of leptin production, the effects of linoleic acid on adipocyte metabolism were also examined. For this purpose, isolated rat adipocytes were incubated with linoleic acid (1-200 microM) in the absence or presence of insulin. Linoleic acid (1-200 microM) significantly decreased insulin-stimulated leptin secretion and expression (P < 0.05), however, no changes in basal leptin production were observed. Linoleic acid also induced a significant decrease (approximately 20%) in adiponectin secretion (P < 0.05), but only in the presence of insulin and at the highest concentration tested (200 microM). This fatty acid did not modify either glucose uptake or lactate production and the percentage of glucose metabolized to lactate was not changed either. Together, these results suggest that linoleic acid seems to interfere with other insulin signalling pathway different from those controlling glucose uptake and metabolism, but involved in the regulation of leptin and adiponectin production.

Conjugated linoleic acid inhibits glucose metabolism, leptin and adiponectin secretion in primary cultured rat adipocytes

Conjugated linoleic acid (CLA) supplementation has been reported to induce insulin resistance in animals and humans, however, the underlying mechanisms remain unclear. The aim of this study was to examine the direct effects of CLA on leptin and adiponectin secretion, two hormones with actions known to influence insulin sensitivity. Isolated rat adipocytes were incubated with CLA (1–200 μM) in the absence and presence of insulin (1.6 nM). CLA inhibited both basal and insulin-stimulated leptin gene expression and secretion (−30 to −40%, P < 0.05–0.01). CLA also inhibited basal adiponectin production (−20 to −40%, P < 0.05–0.01), but not in the presence of insulin. CLA (50–200 μM) decreased basal glucose uptake ( P < 0.05–0.01) and significantly increased the proportion of glucose metabolized to lactate ( P < 0.01). Insulin treatment partially prevented the inhibitory effects of CLA on glucose uptake and induced a significant increase ( P < 0.05–0.01) in the percentage of glucose metabolized to lactate. A strong inverse relationship was observed between the increase in the anaerobic utilization of glucose and the decreases of both leptin and adiponectin secretion. In addition, lipolysis and the expression of the adipogenic transcription factor PPARγ were decreased by CLA. These results indicate that CLA inhibits leptin and adiponectin secretion and suggest that increased anaerobic metabolism of glucose may be involved in these effects. The inhibition of PPARγ could also mediate the inhibition of adiponectin induced by CLA. Furthermore, the inhibition of leptin and adiponectin production induced by CLA may contribute to insulin resistance observed in CLA-treated animals and humans.

Dual Roles of P2 Purinergic Receptors in Insulin-stimulated Leptin Production and Lipolysis in Differentiated Rat White Adipocytes

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP >> UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.

Regulation of leptin secretion from white adipocytes by insulin, glycolytic substrates, and amino acids

The aim of the present study was to determine the respective roles of energy substrates and insulin on leptin secretion from white adipocytes. Cells secreted leptin in the absence of glucose or other substrates, and addition of glucose (5 mM) increased this secretion. Insulin doubled leptin secretion in the presence of glucose (5 mM), but not in its absence. High concentrations of glucose (up to 25 mM) did not significantly enhance leptin secretion over that elicited by 5 mM glucose. Similar results were obtained when glucose was replaced by pyruvate or fructose (both 5 mM). L-Glycine or L-alanine mimicked the effect of glucose on basal leptin secretion but completely prevented stimulation by insulin. On the other hand, insulin stimulated leptin secretion when glucose was replaced by L-aspartate, L-valine, L-methionine, or L-phenylalanine, but not by L-leucine (all 5 mM). Interestingly, these five amino acids potently increased basal and insulin-stimulated leptin secretion in the presence of glucose. Unexpectedly, L-glutamate acutely stimulated leptin secretion in the absence of glucose or insulin. Finally, nonmetabolizable analogs of glucose or amino acids were without effects on leptin secretion. These results suggest that 1) energy substrates are necessary to maintain basal leptin secretion constant, 2) high availability of glycolysis substrates is not sufficient to enhance leptin secretion but is necessary for its stimulation by insulin, 3) amino acid precursors of tricarboxylic acid cycle intermediates potently stimulate basal leptin secretion per se, with insulin having an additive effect, and 4) substrates need to be metabolized to increase leptin secretion.

Eicosapentaenoic fatty acid increases leptin secretion from primary cultured rat adipocytes: role of glucose metabolism

Eicosapentaenoic acid (EPA), one of the n-3 polyunsaturated fatty acids, has been shown to stimulate leptin mRNA expression and secretion in 3T3-L1 cells. However, other studies have reported inhibitory effects of EPA on leptin expression and secretion in vivo and in vitro. To determine the direct effects of EPA on basal and insulin-stimulated leptin secretion, isolated rat adipocytes were incubated with EPA in the absence and presence of insulin. EPA (10, 100, and 200 microM) increased basal leptin gene expression and secretion (+43.8%, P < 0.05; +71.1%, P < 0.01; and +73.7%, P < 0.01, respectively). EPA also increased leptin secretion in the presence of 1.6 nM insulin; however, the effect was less pronounced than in the absence of it. Because adipocyte glucose and lipid metabolism are involved in the regulation of leptin production, the metabolic effects of this fatty acid were also examined. EPA (200 microM) increased basal glucose uptake in isolated adipocytes (+50%, P < 0.05). Anaerobic metabolism of glucose, as assessed by lactate production and proportion of glucose metabolized to lactate, has been shown to be inversely correlated to leptin secretion and was decreased by EPA in both the absence and presence of insulin. EPA increased basal glucose oxidation as determined by the proportion of (14)C-labeled glucose metabolized to CO(2). Lipogenesis ((14)C-labeled glucose incorporation into triglyceride) was decreased by EPA in the absence of insulin, whereas lipolysis (glycerol release) was unaffected. The EPA-induced increase of basal leptin secretion was highly correlated with increased glucose utilization (r = +0.89, P < 0.01) and inversely related to the anaerobic glucose metabolism to lactate. EPA's effect on insulin-stimulated leptin secretion was not related to increased glucose utilization but was inversely correlated with anaerobic glucose metabolism to lactate (r = -0.84, P < 0.01). Together, the results suggest that EPA, like insulin, stimulates leptin production by increasing the nonanaerobic/oxidative metabolism of glucose.

Role of calcium in the secretion of leptin from white adipocytes.

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, (45)Ca 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 (45)Ca 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.

Leptin Effects on Pancreatic β-Cell Gene Expression and Function

The hormone leptin is secreted from white adipocytes, and serum levels of leptin correlate with adipose tissue mass. Leptin was first described to act on the satiety center in the hypothalamus through specific receptors (leptin receptor [ObR]) to restrict food intake and enhance energy expenditure. Important peripheral actions of leptin involve inhibition of insulin biosynthesis and secretion in pancreatic beta-cells. In turn, insulin stimulates leptin secretion from adipose tissue, establishing a hormonal regulatory feedback loop-the so-called "adipo-insular axis." Multiple signal transduction pathways are involved in leptin signaling in pancreatic beta-cells. We have identified the proinsulin gene and protein phosphatase 1 gene as leptin repressed genes and the gene for the suppressor of cytokine signaling 3 protein as a leptin-induced gene in pancreatic beta-cells. The molecular effects of leptin culminate to restrict insulin secretion and biosynthesis to adapt glucose homeostasis to the amount of body fat. In most overweight individuals, however, physiological regulation of body weight by leptin seems to be disturbed, representing "leptin resistance." This leptin resistance at the level of the pancreatic beta-cell may contribute to dysregulation of the adipo-insular axis and promote the development of hyperinsulinemia and manifest type 2 diabetes in overweight patients.

Efectos del ácido araquidónico sobre la secreción y expresión de leptina en cultivos primarios de adipocitos de rata

Leptin, a hormone produced in adipocytes, is a key signal in the regulation of food intake and energy expenditure. Several studies have suggested that leptin can be regulated by macronutrients intake. Arachidonic acid is a dietary fatty acid known to affect cell metabolism. Controversial effects of this fatty acid on leptin have been reported. The aim of this experimental trial was to evaluate the effect of the arachidonic acid on basal and insulin-stimulated leptin secretion and expression in isolated rat adipocytes. Because insulin-stimulated glucose metabolism is an important regulator of leptin expression and secretion by the adipocytes, the effects of the arachidonic acid on indices of adipocyte metabolism were also examined. Isolated adipocytes were incubated with arachidonic acid (1-200 microM) in the absence and presence of insulin (1.6 nM). Leptin secretion and expression, glucose utilization and lactate production were determined at 96 h. The arachidonic acid (200 microM) inhibited both the basal and insulin stimulated leptin secretion and expression. Glucose utilization was not affected by the acid. Basal lactate production was increased by the fatty acid at the highest concentration used (200 microM), however lactate production in presence of insulin was not modified. Finally, the percentage of glucose carbon released as lactate was significantly increased (200 microM). These results suggest that the inhibitory effect of the arachidonic acid on leptin secretion and expression may be due, al least in part, to the increase in the anaerobic utilization of glucose.

Effects of inhibiting transcription and protein synthesis on basal and insulin-stimulated leptin gene expression and leptin secretion in cultured rat adipocytes

We have previously reported that glucose metabolism mediates the effects of insulin to increase leptin gene expression and leptin secretion by isolated adipocytes. The aim of the present study was to investigate the role of transcription and translation in the regulation of basal and insulin-stimulated leptin production. The short-term (4 h) and long-term (24–48 h) effects of actinomycin D, a transcriptional inhibitor, and cycloheximide, an inhibitor of protein synthesis, on leptin gene expression and leptin secretion by isolated adipocytes were determined. Actinomycin D (5 μg/ml) increased both basal and insulin-stimulated (1.6 nM) leptin secretion at 4 and 24 h (193 ± 14.9% and 153.8 ± 10.4% of respective controls at 24 h, both p<0.001). Similar effects of actinomycin D were observed on basal and insulin-stimulated leptin mRNA levels. 5,6-Dichlororibofuranosyl benzimidazole (DRB), another inhibitor of transcription, also increased basal (175.4 ± 18.2% of control; p<0.01) and insulin-stimulated leptin secretion (141.0 ± 11.1% of insulin-treated cells; p<0.05) at 24 h. The effect of actinomycin D and DRB to increase basal leptin secretion observed at 4 and 24 h was not present at 48 h when actinomycin D and DRB both markedly inhibited insulin-stimulated leptin secretion (to 36 ± 16%, p<0.05 and 21.9 ± 5.6% of control, for actinomycin D and DRB, respectively, both p<0.001). Neither actinomycin D nor DRB had any effect on adipocyte glucose utilization between 24 and 48 h. The observed effects of inhibitors of transcription on leptin gene expression and leptin secretion are consistent with a long-term transcriptional mechanism for insulin-stimulated glucose metabolism to increase leptin production. Cycloheximide treatment (10 μg/ml) abolished the effects of insulin to stimulate leptin secretion (29 ± 11% of control, p<0.01) during the first 4 h of treatment and at all later time points, which indicate that de novo protein synthesis is required for insulin-mediated glucose metabolism to increase leptin secretion.

Regulation of leptin secretion from white adipocytes by free fatty acids.

Norepinephrine stimulates lipolysis and concurrently inhibits insulin-stimulated leptin secretion from white adipocytes. To assess whether there is a cause-effect relationship between these two metabolic events, the effects of fatty acids were investigated in isolated rat adipocytes incubated in buffer containing low (0.1%) and high (4%) albumin concentrations. Palmitic acid (1 mM) mimicked the inhibitory effects of norepinephrine (1 microM) on insulin (10 nM)-stimulated leptin secretion, but only at low albumin concentrations. Studies investigating the effects of the chain length of saturated fatty acids [from butyric (C4) to stearic (C18) acids] revealed that only fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion. Long-chain mono- and polyunsaturated fatty acids constitutively present in adipocyte triglyceride stores (oleic, linoleic, gamma-linolenic, palmitoleic, eicosapentanoic, and docosahexanoic acids) also completely suppressed leptin secretion. Saturated and unsaturated fatty acids inhibited insulin-stimulated leptin secretion with the same potency and without any significant effect on basal secretion. On the other hand, inhibitors of mitochondrial fatty acid oxidation (palmoxirate, 2-bromopalmitate, 2-bromocaproate) attenuated the stimulatory effects of insulin on leptin release without reversing the effects of fatty acids or norepinephrine, suggesting that fatty acids do not need to be oxidized by the mitochondria to inhibit leptin release. These results demonstrate that long-chain fatty acids mimic the effects of norepinephrine on leptin secretion and suggest that they may play a regulatory role as messengers between stimulation of lipolysis by norepinephrine and inhibition of leptin secretion.

Circulating leptin response to feeding and exogenous infusion of insulin in sheep exposed to thermoneutral and cold environments

Leptin has been shown to regulate feed intake and energy expenditure. Insulin stimulates leptin secretion in rodents, but its action on leptin secretion is still obscure in ruminants. If insulin stimulates leptin secretion in ruminants, circulating leptin concentrations may change during exposure to cold, because of fluctuating insulin secretion and action in the cold environment. The present experiment was designed to determine whether feeding or exogenous administration of insulin affects circulating leptin levels in sheep exposed to thermoneutral and cold environments. Suffolk rams that were shorn and fed a diet once daily were subjected to a thermoneutral (20 °C) or cold (0 °C) environment for at least 1 week. Overall mean concentrations of plasma leptin in the feeding experiment were lower ( P<0.05) in the cold environment than in the thermoneutral environment. Plasma leptin levels remained relatively unchanged after feeding in both environments, though plasma insulin response to feeding in both environments increased ( P<0.01). The euglycemic clamps (insulin infusion rate: 4 mU kgBW −1 min −1 for 2 h) increased ( P<0.01) circulating leptin concentrations in the thermoneutral, but not in the cold environment. These results suggest that lower circulating leptin levels in ruminants exposed to the cold environment could be partly due to the depressed insulin action on leptin secretion.

Mechanisms of leptin secretion from white adipocytes.

The mechanisms regulating leptin secretion were investigated in isolated rat white adipocytes. Insulin (1-100 nM) linearly stimulated leptin secretion from incubated adipocytes for at least 2 h. The adrenergic agonists norepinephrine, isoproterenol (two nonselective beta-agonists), or CL-316243 (potent beta3) all inhibited insulin (10 nM)-stimulated leptin release. The inhibitory effects of norepinephrine and isoproterenol could be reversed not only by the nonselective antagonist propranolol but also by the selective antagonists ICI-89406 (beta1) or ICI-118551 (beta2), the beta2-antagonist being less effective than the beta1. Insulin-stimulated leptin secretion could also be inhibited by a series of agents increasing intracellular cAMP levels, such as lipolytic hormones (ACTH and thyrotropin-stimulating hormone), various nonhydrolyzable cAMP analogs, pertussis toxin, forskolin, methylxanthines (caffeine, theophylline, IBMX), and specific inhibitors of phosphodiesterase III (imazodan, milrinone, and amrinone). Significantly, antilipolytic agents other than insulin (adenosine, nicotinic acid, acipimox, and orthovanadate) did not mimic the acute stimulatory effects of insulin on leptin secretion under these conditions. We conclude that norepinephrine specifically inhibits insulin-stimulated leptin secretion not only via the low-affinity beta3-adrenoceptors but also via the high-affinity beta1/beta2-adrenoceptors. Moreover, it is suggested that 1) activation of phosphodiesterase III by insulin represents an important metabolic step in stimulation of leptin secretion, and 2) lipolytic hormones competitively counterregulate the stimulatory effects of insulin by activating the adenylate cyclase system.

Effects of Trecadrine, a beta3-adrenergic agonist, on leptin secretion, glucose and lipid metabolism in isolated rat adipocytes.

Leptin, a hormone produced in adipocytes, is a key signal in the regulation of food intake and energy expenditure. Beta-adrenergic agonists have been shown to inhibit leptin gene expression and leptin secretion. The mechanisms underlying the inhibitory effects of beta-adrenergic agonists have not been established. In this study, we examined the effects of Trecadrine, a novel beta3-adrenergic agonist, on basal and insulin-stimulated leptin secretion in isolated rat adipocytes. Because insulin-stimulated glucose metabolism is an important regulator of leptin expression and secretion by the adipocytes, the effects of Trecadrine on indices of adipocyte metabolism were also examined. Isolated adipocytes were incubated with Trecadrine (10(-8)-10(-4) M) in the absence or presence of insulin (1.6 nM). Leptin secretion, glucose utilization, lactate production, glucose incorporation into CO(2) and triglyceride, as well as lipolysis (glycerol release) were determined. Trecadrine induced a concentration-dependent inhibition of basal leptin secretion. Trecadrine also decreased insulin-stimulated leptin secretion; however, the effect was not as pronounced as in the absence of insulin. Treatment of adipocytes with Trecadrine increased basal glucose utilization and produced a further increase in insulin-stimulated glucose utilization. Basal lactate production was also increased by Trecadrine; however, the proportion (percentage) of glucose carbon released as lactate was unaffected. In the presence of insulin, absolute lactate production was unaffected by Trecadrine at 96 h. However, the percentage of glucose carbon released as lactate was significantly decreased by insulin treatment, and was further decreased by the co-treatment with Trecadrine. Trecadrine induced a dose-dependent increase of the absolute amount of glucose incorporated into triglyceride. However, the percentage of glucose utilized that was incorporated into triglyceride was unaffected by Trecadrine. Trecadrine did not modify the proportion of glucose utilized that was oxidized to CO(2). Trecadrine increased glycerol release after 96 h of treatment. Glycerol release was negatively correlated with leptin secretion. These results suggest that alterations of glucose metabolism are not directly involved in the effects of beta3-adrenergic agonists to inhibit leptin expression and secretion. The inverse relationship between leptin secretion and the increase of glycerol levels, which is an index of the activation of cAMP-dependent protein kinases, suggests that activation of the cAMP signaling pathway mediates the inhibitory effects of Trecadrine on leptin gene expression and secretion.

Effects of a β3-Adrenergic Agonist on Glucose Uptake and Leptin Expression and Secretion in Cultured Adipocytes from Lean and Overweight (Cafeteria) Rats

The increase in body and white adipose tissue weights induced by a high-fat diet were prevented by treatment with the β3-adrenergic agonist Trecadrine. Plasma insulin levels were slightly elevated in overweight rats, while a decrease was observed in Trecadrine-treated groups. Insulin-dependent glucose uptake was impaired in adipocytes of the overweight rats in relation to lean animals. The β3-adrenergic agonist induced an increase in insulin-stimulated glucose uptake by adipocytes as compared to the nontreated animals. In fact, Trecadrine treatment was able to restore to control values the impairment in insulin-mediated glucose uptake induced by the cafeteria diet, suggesting that Trecadrine prevents the development of insulin resistance in overweight animals. Basal leptin secretion was increased in adipocytes of the overweight rats in relation to lean animals. Trecadrine treatment induced a decrease in basal leptin secretion compared to the untreated animals. Insulin-stimulated leptin secretion reached similar levels in adipocytes of the overweight rats as in lean animals. There was a trend for insulin-induced leptin secretion to be lower at 24 h in Trecadrine-treated rats, but it did not reach statistical significance. In conclusion, adipocytes of diet-induced overweight animals have a higher basal leptin secretion, which is reduced by treatment with Trecadrine. However, neither the cafeteria diet nor the Trecadrine treatment significantly alters the ability of adipocytes to increase leptin secretion in response to insulin.

Insulin and glucocorticoids differentially regulate leptin transcription and secretion in brown adipocytes.

Leptin, the ob gene product, is produced by adipose tissue and is submitted to a complex hormonal and metabolic regulation. Leptin plays a critical role in the balance of body weight. Here we report on secretion and hormonal regulation of leptin by brown adipocytes. Using the recently established T37i cell line, we show that leptin expression and secretion occurred as a function of cell differentiation. In differentiated T37i cells, insulin induced leptin release ( approximately 0.25 ng/10(6) cells/h) in a concentration-dependent manner (EC50=0.1 nM), and this was totally suppressed by beta3-adrenergic ligand, thiazolidinedione, cycloheximide, or actinomycin D. Insulin induced a strong, rapid (within 2 h) but transient fivefold increase in leptin mRNA levels. This transcriptional control of ob gene expression by insulin involved both phosphatidylinositol 3-kinase- and MAP kinase-dependent pathways. Glucocorticoids inhibited both insulin-stimulated leptin secretion and ob gene expression without affecting leptin mRNA stability (t(1/2)=3h05). Altogether, our results demonstrate that brown adipocytes express and secrete leptin, whose hormonal regulation clearly differs from that described in white adipose tissue. These findings point to tissue-specific molecular mechanisms and suggest that leptin might exert direct effects on energy homeostasis through an autocrine mechanism.