Stimulation by eicosapentaenoic acids of leptin mRNA expression and its secretion in mouse 3T3-L1 adipocytes in vitro

Recent evidence indicates that both leptin and eicosapentaenoic acids (EPA) improve insulin sensitivity. In the present study, we examined the effect of EPA on endogenous leptin expression in 3T3-L1 adipocytes to clarify whether the EPA's effect is exerted through leptin expression. EPA caused a time- and dose-dependent increase of leptin mRNA levels in 3T3-L1 adipocytes. Leptin mRNA expression was significantly increased up to 309.4 +/- 17.0% of the control by 24 h (P < 0.01; n = 6). Leptin secretion was also significantly increased up to 193.3 +/- 12.1% of the control by 24 h (P < 0.01; n = 6). EPA is a ligand for peroxisome proliferator-activated receptors (PPARs) with the highest affinity to PPARalpha. We examined the effect on leptin expression of clofibrate, a ligand for PPARalpha, bezafibrate, for PPARbeta, or troglitazone, for PPARgamma, to clarify whether these ligands for PPARs could mimic EPA-induced stimulation of leptin expression. Neither clofibrate nor bezafibrate affected leptin mRNA expression, whereas troglitazone significantly suppressed leptin mRNA expression. On the other hand, inhibition by 6-diazo-5-oxo-l-norleucine of the rate-limiting enzyme in hexosamine biosynthesis blunted EPA-induced stimulation of leptin mRNA expression and its secretion. These data suggest that EPA up-regulates leptin gene expression and its secretion probably through a hexosamine biosynthetic pathway.     

Control of proteolysis by norepinephrine and insulin in brown adipocytes: role of ATP,phosphatidylinositol 3-kinase,and p70 S6K

The objective of this study was to evaluate some of the mechanisms by which norepinephrine (NE) and insulin may influence protein degradation in mouse brown adipocytes differentiated in cultures. The effects of NE and insulin, alone or in combination, on three factors known to influence proteolysis (maintenance of cell ATP and 1-phosphatidylinositol 3-kinase (PI 3-kinase) and p70 ribosomal S6-kinase (p70 S6K) activities) were examined. It was proposed that NE affects proteolysis indirectly by decreasing cell ATP from activation of uncoupling protein-1 (UCP1)-dependent mitochondrial respiration. This was tested by comparing the effects of NE and fatty acids (which directly activate UCP1) on proteolysis in brown adipocytes, as well as in pre-adipocytes and 3T3-L1 adipocytes, which do not express UCP1. An inhibitory effect of insulin on proteolysis is observed in both pre-adipocytes and differentiated cells, whereas NE and exogenously added fatty acids inhibit proteolysis only in brown adipocytes. There is a linear relationship between reductions in cell ATP and proteolysis in response to increasing concentrations of NE or fatty acids. PI 3-kinase activity is required for proteolysis, because two selective inhibitors (wortmannin and LY294002) reduce proteolysis in both pre-adipocytes and differentiated cells. This effect is not additive to that of NE, which suggests they affect the same proteolytic pathway. In contrast to NE, insulin increases PI 3-kinase activity and phosphorylation of p70 S6K. Rapamycin, which prevented insulin-dependent increase in phosphorylation of p70 S6K, increases proteolysis in brown adipocytes and antagonizes the inhibitory effect of insulin on proteolysis, but not the inhibitory effect of NE. Thus, insulin inhibits proteolysis via rapamycin-sensitive activation of p70 S6K, whereas the effect of NE appears largely to be a function of decreasing cell ATP content.     

Nitric oxide-induced downregulation of leptin production by 3T3-L1 adipocytes.

Leptin secreted mainly by adipocytes plays an important role in insulin sensitivity in metabolic syndrome. Inducible nitric oxide synthase (iNOS) in 3T3-L1 adipocytes is induced by lipopolysaccharide (LPS) and several proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma (IFN-gamma). Because the role of iNOS-derived nitric oxide (NO) in adipocyte function has not been fully clarified, the question that we addressed in the present study was whether iNOS-derived NO is involved in regulation of leptin secretion by adipocytes. Incubation of 3T3-L1 adipocytes for 12h with a mixture of IFN-gamma and LPS caused not only a 55% reduction in leptin secretion and a 52% reduction in leptin mRNA, but also significant induction of iNOS at both protein and mRNA levels. Inhibition of leptin secretion that had been induced by the IFN-gamma-LPS mixture was completely nullified by NOS inhibitors such as Nomega-monomethyl-L-arginine and aminoguanidine. Treatment of adipocytes with NO donors such as an NONOate and S-nitrosoglutathione produced an effect on leptin secretion similar to that of the IFN-gamma-LPS mixture. It is likely therefore that NO mediates downregulation of leptin caused by the IFN-gamma-LPS mixture in 3T3-L1 adipocytes, which suggests an important role for NO in adipocyte functions.     

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.     

Novel form of lipolysis induced by leptin.

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nM norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p < 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p < 0.05). NE (100 nM) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARalpha, ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARalpha and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.     

Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice

Adiponectin (ApN) and leptin are two adipocytokines that control fuel homeostasis, body weight, and insulin sensitivity. Their interplay is still poorly studied. These hormones are either undetectable or decreased in obese, diabetic ob/ob mice. We examined the effects of leptin treatment on ApN gene expression, protein production, secretion, and circulating levels of ob/ob mice. We also briefly tackled the influence of this treatment on resistin, another adipocytokine involved in obesity-related insulin resistance. Leptin-treated (T) obese mice (continuous sc infusion for 6 days) were compared with untreated lean (L), untreated obese (O), and untreated pair-fed obese (PF) mice. Blood was collected throughout the study. At day 3 or day 6, fat pads were either directly analyzed (mRNA, ApN content) or cultured for up to 24 h (ApN secretion). The direct effect of leptin was also studied in 3T3-F442A adipocytes. Compared with L mice, ApN content of visceral or subcutaneous fat and ApN secretion by adipose explants were blunted in obese mice. Accordingly, plasma ApN levels of O mice were decreased by 50%. Leptin treatment of ob/ob mice increased ApN mRNAs, ApN content, and secretion from the visceral depot by 50-80%. Leptin also directly stimulated ApN mRNAs and secretion from 3T3-F442A adipocytes. After 6 days of treatment, plasma ApN of ob/ob mice increased 2.5-fold, a rise that did not occur in PF mice. Plasma resistin of T mice was barely decreased. Leptin treatment, but not mere calorie restriction, corrects plasma ApN in obese mice by restoring adipose tissue ApN concentrations and secretion, at least in part, via a direct stimulation of ApN gene expression. Such a treatment only minimally affects circulating resistin. ApN restoration could, in concert with leptin, contribute to the metabolic effects classically observed during leptin administration.     

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 Regulates KATP Channel Trafficking in Pancreatic β-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)

Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (K_ATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing K_ATP channel conductance in β-cells. We investigated the mechanism by which leptin increases K_ATP channel conductance. We show that leptin causes a transient increase in surface expression of K_ATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on K_ATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on K_ATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates K_ATP channel trafficking to modulate β-cell function and insulin secretion.     

Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes

Adiponectin and leptin are two adipokines secreted by white adipose tissue that regulate insulin sensitivity. Previously we reported that adiponectin but not leptin release depends on GGA-coated vesicle formation, suggesting that leptin and adiponectin may follow different secretory routes. Here we have examined the intracellular trafficking pathways that lead to the secretion of these two hormones. While adiponectin and leptin displayed distinct localization in the steady-state, treatment of adipocytes with brefeldin A inhibited both adiponectin and leptin secretion to a similar level, indicating a common requirement for class III ADP-ribosylating factors and an intact Golgi apparatus. Adiponectin secretion was significantly reduced by endosomal inactivation in both 3T3L1 and rat isolated adipocytes, whereas this treatment had no effect on leptin secretion. Importantly, endosomal inactivation completely abolished the insulin stimulatory effect on adiponectin release in rat adipocytes. Confocal microscopy studies revealed colocalization of adiponectin with endogenous rab11 a marker for the recycling endosome, and with expressed rab5-GFP mutant (rab5Q75L) a marker for the early endosome compartment. Colocalization of adiponectin and rab5Q75L was increased in endosome inactivated cells. Consistent with these findings adiponectin secretion was reduced in cells expressing mutants of Rab11 and Rab5 proteins. In contrast, expression of an inactive (kinase dead) mutant of Protein Kinase D1 moderately but significantly inhibited leptin secretion without altering adiponectin secretion. Taken together, these results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and that endosomal compartments are required for adiponectin but not for leptin secretion.     

Direct and indirect effects of leptin on adipocyte metabolism

Leptin is hypothesized to function as a negative feedback signal in the regulation of energy balance. It is produced primarily by adipose tissue and circulating concentrations correlate with the size of body fat stores. Administration of exogenous leptin to normal weight, leptin responsive animals inhibits food intake and reduces the size of body fat stores whereas mice that are deficient in either leptin or functional leptin receptors are hyperphagic and obese, consistent with a role for leptin in the control of body weight. This review discusses the effect of leptin on adipocyte metabolism. Because adipocytes express leptin receptors there is the potential for leptin to influence adipocyte metabolism directly. Adipocytes also are insulin responsive and receive sympathetic innervation, therefore leptin can also modify adipocyte metabolism indirectly. Studies published to date suggest that direct activation of adipocyte leptin receptors has little effect on cell metabolism in vivo, but that leptin modifies adipocyte sensitivity to insulin to inhibit lipid accumulation. In vivo administration of leptin leads to a suppression of lipogenesis, an increase in triglyceride hydrolysis and an increase in fatty acid and glucose oxidation. Activation of central leptin receptors also contributes to the development of a catabolic state in adipocytes, but this may vary between different fat depots. Leptin reduces the size of white fat depots by inhibiting cell proliferation both through induction of inhibitory circulating factors and by contributing to sympathetic tone which suppresses adipocyte proliferation. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Endothelin-1 stimulates leptin production in adipocytes

Leptin is an adipocyte-derived hormone that regulates body fat stores and feeding behavior. In an effort to identify endogenous diffusible modulators of leptin production, we found that endothelin-1 (ET-1) up-regulates leptin expression in adipocytes. ET-1 is as potent and efficacious as insulin in stimulating leptin production in two different adipocyte cell lines. Endothelins stimulate leptin production via the endothelin-A receptor (ET(A)), as judged by a potency rank order of ET-1 ET-3. We detected expression of ET(A) but not ET(B) in both cell lines by Northern blot analysis. In addition, the ET(A)-selective antagonist FR139317 inhibited ET-1-induced leptin expression more potently than did the ET(B)-selective antagonist BQ788. ET-1 and insulin positively interact with each other in increasing leptin production in adipocytes. In primary mouse white fat cells, we detected expression of both ET(A) and ET(B) by Northern blot and in situ hybridization analyses. We conclude that ET-1 stimulates leptin production via the ET(A) receptor in cultured adipocytes.     

Potential role of leptin in endochondral ossification.

Leptin, a 16-kD circulating hormone secreted mainly by white adipose tissue, is a product of the obese (ob) gene. Leptin acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes. Leptin also inhibits bone formation through a hypothalamic relay. To obtain a better understanding of the potential role of leptin in bone formation, the localization of leptin in endochondral ossification was examined immunohistochemically. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage and in a number of osteoblasts of the primary spongiosa beneath the growth plate. The hypertrophic chondrocytes far from the blood vessels were negative for leptin. Moreover, we detected the production and secretion of leptin by a mouse osteoblast cell line (MC3T3-E1) and a mouse chondrocyte cell line (MCC-5) by RT-PCR, immunocytochemistry, and Western blotting. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells (HUVECs) in vitro. These findings suggest the possibility that leptin exerts its influence on endochondral ossification by regulating angiogenesis.