Genistein restricts leptin secretion from rat adipocytes

The isoflavones--genistein and daidzein -- compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein-- an analogue of genistein -- used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2h in Krebs--Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25--1mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.     

Effects of arachidonic acid on leptin secretion and expression in primary cultured rat adipocytes

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.     

T3 and Triac inhibit leptin secretion and expression in brown and white rat adipocytes

Leptin regulates appetite, inhibits food intake, and seems to increase energy expenditure. We investigated the effect of triiodothyroacetic acid (Triac), a metabolite of T3, which seems to be more thermogenic than T3, on leptin secretion and mRNA expression. Rat primary cultures of white and brown adipocytes were treated with increasing concentrations of Triac and T3. The effect of different types of serum and insulin concentrations was also tested. Serum inhibited leptin secretion and mRNA expression. Leptin secretion was also clearly inhibited by Triac and T3 in a dose-dependent manner and with similar potency. In the presence of norepinephrine (NE), Triac and T3 had a similar inhibitory effect, but the inhibition was almost complete in white adipocytes. Parallel results were found at the mRNA level, where Triac and T3 had similar inhibitory potency, both alone and with NE. We also show that insulin induced dose- and time-dependent increases in leptin secretion, reaching maximum levels at 0.5 and 3 nM insulin for white and brown adipocytes, respectively. Leptin secretion was higher in white than in brown adipocytes. The increases in leptin secretion were preceded by increases in leptin mRNA. In conclusion, these data demonstrate for the first time that Triac, like T3 and serum, inhibits leptin secretion and expression in white and brown adipocytes, whereas insulin has the opposite effect.     

Anthocyanin enhances adipocytokine secretion and adipocyte-specific gene expression in isolated rat adipocytes

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte-specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. In this study, we demonstrated that anthocyanins (cyanidin or cyanidin 3-glucoside) have the potency of a unique pharmacological function in isolated rat adipocytes. Treated adipocytes with anthocyanins enhanced adipocytokine (adiponectin and leptin) secretion and up-regulated the adipocyte specific gene expression without activation of PPARgamma in isolated rat adipocytes. The gene expression of adiponectin was also up-regulated in white adipose tissue in mice fed an anthocyanin supplemented diet. As one of the possible mechanisms, AMP-activated protein kinase activation would be associated with these changes, nevertheless, the AMP:ATP ratio was significantly decreased by administration of the anthocyanins. These data suggest that anthocyanins have a potency of unique therapeutic advantage and also have important implications for preventing obesity and diabetes.     

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.     

Melatonin enhances leptin expression by rat adipocytes in the presence of insulin

Leptin and melatonin play an important role in the regulation of body mass and energy balance. Both hormones show a circadian rhythm, with increasing values at night. In addition, melatonin receptors were recently described in adipocytes, where leptin is synthesized. Here, we investigated the influence of melatonin and its interaction with insulin and dexamethasone on leptin expression. Isolated rat adipocytes were incubated with melatonin (1 nM) alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) for 6 h. Melatonin or insulin alone did not affect leptin expression, but together they increased it by 120%. Dexamethasone increased leptin mRNA content (105%), and this effect was not enhanced by melatonin. Simultaneous treatment with the three hormones provoked a further increase in leptin release (250%) and leptin mRNA (100%). Melatonin prevented the forskolin-induced inhibition (95%) of leptin expression. In addition, melatonin's ability to stimulate leptin release (in the presence of insulin) was completely blocked by pertussis toxin and luzindole. To gain further insight into the molecular basis of melatonin and insulin synergism, the insulin-signaling pathway was investigated. Melatonin increased the insulin-induced insulin receptor-beta tyrosine phosphorylation, which led to an increased serine phosphorylation of the downstream convergent protein Akt. We concluded that melatonin interacts with insulin and upregulates insulin-stimulated leptin expression. These effects are caused by melatonin binding to the pertussis toxin-sensitive G(i) protein-coupled membrane receptor (MT1 subtype) and the cross talk with insulin, since insulin receptor and its convergent target Akt are coactivated by melatonin.     

Intracellular mediators in regulation of leptin secretion from adipocytes

Leptin is a hormone primarily secreted by adipocytes and participating in the regulation of food intake and energy expenditure. Its blood levels usually correlate with adiposity. The secretion of this hormone is affected, among others, by food consumption, insulin, fasting and cold exposure. Regulation of leptin secretion depends on many intracellular events. It is known that the activation of mTOR (the mammalian target of rapamycin) as well as increase in ATP and malonyl-CoA content in adipocytes enhance secretion of leptin. The rise in intracellular cAMP and fatty acids is thought to evoke the opposite effect. Moreover, the undisturbed action of endogenous adenosine in adipocytes and the proper intracellular Ca(2+) concentration in these cells were also found to have an important function in leptin release. The role of mTOR, ATP, cAMP, fatty acids, malonyl-CoA, adenosine and Ca(2+) in the regulation of leptin secretion from adipocytes is discussed.     

Regulation of metallothionein gene expression and secretion in rat adipocytes differentiated from preadipocytes in primary culture.

The gene encoding metallothionein, a low mol. wt. metal binding and stress response protein, is expressed in white adipose tissue. In the present study, metallothionein (MT-1) gene expression and factors regulating metallothionein production have been examined in adipocytes induced to differentiate from fibroblastic preadipocytes in primary cell culture. On the induction of differentiation, the metallothionein-1 gene was strongly expressed in the cells and metallothionein released into the medium. A peak in metallothionein-1 mRNA level and metallothionein secretion occurred at 2 and 10 days post-differentiation, respectively, with a decrease in protein release after this time. The metallothionein-1 gene was expressed in the adipocytes prior to the adipsin and lipoprotein lipase genes, suggesting that it is an early marker of adipocyte differentiation. The addition of the glucocorticoid, dexamethasone, led to a substantial increase in metallothionein-1 mRNA in the cells and metallothionein secretion. Insulin and leptin also stimulated metallothionein production, although the effect was small. Neither noradrenaline nor the beta3-adrenoceptor agonist, BRL 37 344, altered metallothionein release but forskolin and bromo-cAMP were stimulatory, markedly increasing both metallothionein-1 level and metallothionein secretion. It is suggested that metallothionein is a novel secretory product of the differentiated white adipocyte and that its production is regulated particularly by glucocorticoids and through a cAMP-dependent pathway.     

Ischemia/reperfusion in rat heart induces leptin and leptin receptor gene expression

Concentrations of leptin, an adipocyte-derived hormone, are elevated in obesity. Recently, leptin has been shown to participate in multiple biological actions including inflammation, reproduction, and angiogenesis. Leptin has also been documented as a critical component in the process of wound healing; however, leptin involvement in cardiovascular disease is poorly understood. We examined the expression of leptin (ob) and leptin receptor (ob-R) genes in the rat heart following ischemia/reperfusion, which was induced by coronary artery ligation, and mRNA was obtained from hearts 0.5 to 36 h after initiating reperfusion. Expressions of ob and ob-R mRNA were examined by real-time quantitative RT-PCR and immunohistochemistry. The ob and ob-Ra mRNA and protein expressions were significantly increased (p<0.01) and ob-Rb mRNA was significantly decreased (p<0.01) in hearts after 8 h of reperfusion. Furthermore, ob and ob-R proteins were expressed in injured myocytes where inflammatory cells infiltrated. In contrast, those expressions were not influenced in hearts after 8 h of ischemia stress only. To determine the functional effects of leptin on the ischemic/reperfused heart, rats were treated with anti-leptin antibodies prior to ischemia/reperfusion; however, this treatment did not affect the elevation of mRNA expression levels of inflammatory markers such as TNF-alpha and IL-1beta in ischemic hearts. Our results demonstrated for the first time that ischemia/reperfusion induced leptin and leptin receptor gene expression in the rat heart. This study helps to elucidate the mechanisms behind the onset and development of ischemic heart disease concomitant with obesity.     

Molecular cloning,expression, and regulation of goose leptin receptor gene in adipocytes

A full-length cDNA encoding the goose (Anser anser) leptin receptor (LEPR) was cloned and sequenced. The goose LEPR gene encodes a 1,156-amino acid protein containing a signal peptide, a single transmembrane domain and specific motifs involving putative leptin-binding and signal transduction. The deduced goose LEPR protein shows more than 90% identity to duck and 75% identity to chicken and turkey. Quantitative real-time analysis reveals that the goose LEPR is predominantly expressed in brain. The expression of LEPR in goose adipocytes can be up-regulated by oleic acid in vitro. Moreover, the expression levels of genes, which have been demonstrated to be related to adipocyte differentiation, are down-regulated in LEPR-knockdown adipocytes, indicating LEPR's potential role in adipocyte differentiation in goose.     

Neuropeptide B and W regulate leptin and resistin secretion, and stimulate lipolysis in isolated rat adipocytes

Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.     

Regulation of adiponectin and leptin secretion and expression by insulin through a PI3K-PDE3B dependent mechanism in rat primary adipocytes

Adiponectin is intimately involved in the regulation of insulin sensitivity, carbohydrate and lipid metabolism, and cardiovascular functions. The circulating concentration of adiponectin is decreased in obesity and Type 2 diabetes. The present study attempts to elucidate the mechanisms underlying the regulation of adiponectin secretion and expression in rat primary adipocytes. The beta-agonist, isoprenaline, decreased adiponectin secretion and expression in a dose-dependent manner in primary adipocytes. Importantly, such an inhibitory effect could be blocked by insulin. The opposing effects of isoprenaline and insulin could be explained by differential regulation of intracellular cAMP levels, since cAMP analogues suppressed adiponectin secretion and expression in a fashion similar to isoprenaline, and insulin blocked the inhibitory effects of the cAMP analogue hydrolysable by PDE (phosphodiesterase). A specific PDE3 inhibitor, milrinone, and PI3K (phosphoinositide 3-kinase) inhibitors abolished the effects of insulin on adiponectin secretion and expression. In the same studies, leptin secretion and expression displayed a similar pattern of regulation to adiponectin. We conclude that insulin and beta-agonists act directly at the adipocytes in opposing fashions to regulate the production of adiponectin and leptin, and that a PI3K-PDE3B-cAMP pathway mediates the effects of insulin to restore beta-agonist/cAMP-suppressed secretion and expression of these two adipokines.     

Leptin expression and leptin receptor gene polymorphisms in growth hormone deficiency patients

Growth hormone deficiency (GHD) patients have lower weight, height, bone age, insulin-like growth factor 1 (IGF-1) levels, GH levels, fat metabolism and skeletal growth. The association of leptin with GHD characteristics and the effect of gene variants of leptin on GHD are unknown. Our aim was to examine the association of circulating leptin levels and common genetic variants in leptin (LEP) and leptin receptor (LEPR) genes with anthropometric measures, circulating hormone concentrations and GHD. A case control study of 125 GHD cases and 159 control subjects were characterized for bone age, body mass index (BMI), height, weight, leptin, IGF-1, GH and their genotype at the leptin promoter G-2548A, and LEPR variants, K109R and Q223R, at Chung Shan Medical University Hospital. Leptin levels were significantly associated with lower bone age, weight and BMI in GHD patients. Leptin levels were also significantly associated with reduced IGF-1 levels in girls but not boys in both groups. The frequency of LEPR223 [A/G or A/A] genotype was significantly higher than the LEPR223 G/G genotype in the GHD group. The LEPR223 [A/G or A/A] genotype was significantly associated with increased weight and BMI in the control group, but not in the GHD group. In conclusion, the GHD group carried a significantly higher frequency of the LEPR [G/A or A/A] genotype and of the A allele (LEPR223R). The LEPR223R polymorphism affected weight and BMI in control, but not in GHD patients, suggesting that the effect of LEPR223 [A/G or A/A] genotype was counteracted by other factor(s) in GHD patients.     

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.     

trans-10,cis-12-Conjugated linoleic acid reduces leptin secretion from 3T3-L1 adipocytes

The trans10,cis12 (t10c12) isomer of conjugated linoleic acid (CLA) has been shown to inhibit heparin-releasable lipoprotein lipase activity, reduce lipid stores in cultured 3T3-L1 adipocytes, and, when fed to mice, reduce body fat gain. We now report that t10c12 CLA significantly reduced leptin secretion from cultured 3T3-L1 adipocytes, and reduced leptin mRNA levels within the cells. Similar effects were produced by conjugated nonadecadienoic acid (a 19-carbon CLA cognate that is more effective than CLA in reducing body fat gain in mice), the lipoxygenase inhibitor nordihydroguaiaretic acid (which is synergistic with CLA in reducing body fat gain in mice), and ciglitazone (TZD, a PPARgamma agonist). Feeding mice diet supplemented with 0.5% t10c12 CLA for 4 weeks significantly reduced body fat gain, serum leptin levels and adipocyte leptin mRNA expression, without affecting feed intake or body weight. These data provide new insights into apparent mechanistic similarities among t10c12 CLA, CNA, NDGA, and TZD.     

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.     

Reduction of leptin secretion by soy isoflavonoids in murine adipocytes in vitro

Daidzein and genistein are the main aglycones of soy isoflavonoid, and have many useful activities in vitro and in vivo. However, equol, a metabolite of daidzein in vivo, has attracted attention due to its stronger activity than that of the naturally occurring isoflavonoids. We subjected the soy isoflavonoids, including the naturally occurring (S)-equol, to mouse adipocytes, and compared the inhibitory activity on the leptin secretion. Equol, daidzein and genistein inhibited the leptin secretion, whereas O-desmethylangolensin had a lower activity. The inhibitory activity of the isoflavones was not affected by the addition of an iNOS inhibitor and an estrogen.