Adipocytes promote prostate cancer stem cell self-renewal through amplification of the cholecystokinin autocrine loop

Obesity has long been linked with prostate cancer progression, although the underlying mechanism is still largely unknown. Here, we report that adipocytes promote the enrichment of prostate cancer stem cells (CSCs) through a vicious cycle of autocrine amplification. In the presence of adipocytes, prostate cancer cells actively secrete the peptide hormone cholecystokinin (CCK), which not only stimulates prostate CSC self-renewal, but also induces cathepsin B (CTSB) production of the adipocytes. In return, CTSB facilitates further CCK secretion by the cancer cells. More importantly, inactivation of CCK receptor not only suppresses CTSB secretion by the adipocytes, but also synergizes the inhibitory effect of CTSB inhibitor on adipocyte-promoted prostate CSC self-renewal. In summary, we have uncovered a novel mechanism underlying the mutual interplay between adipocytes and prostate CSCs, which may help explaining the role of adipocytes in prostate cancer progression and provide opportunities for effective intervention.

Resveratrol Metabolites Modify Adipokine Expression and Secretion in 3T3-L1 Pre-Adipocytes and Mature Adipocytes

Objective: Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. Methods: 3T3-L1 maturing pre-adipocytes were treated during differentiation with 25 mu M of resveratrol or with its metabolites and 3T3-L1 mature adipocytes were treated for 24 hours with 10 mM resveratrol or its metabolites. The gene expression of adiponectin, leptin, visfatin and apelin was assessed by Real Time RT-PCR and their concentration in the incubation medium was quantified by ELISA. Results: Resveratrol reduced mRNA levels of leptin and increased those of adiponectin. It induced the same changes in leptin secretion. Trans-resveratrol-3-O-glucuronide and trans-resveratrol-4'-O-glucuronide increased apelin and visfatin mRNA levels. Trans-resveratrol-3-O-sulfate reduced leptin mRNA levels and increased those of apelin and visfatin. Conclusions: The present study shows for the first time that resveratrol metabolites have a regulatory effect on adipokine expression and secretion. Since resveratrol has been reported to reduce body-fat accumulation and to improve insulin sensitivity, and considering that these effects are mediated in part by changes in the analyzed adipokines, it may be proposed that resveratrol metabolites play a part in these beneficial effects of resveratrol.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]iodophenyl]2-ethoxy propanoic acid], which is specific for the ? isoform of the peroxisomal proliferator activated receptor (PPAR?), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPAR?1 and to a GST (glutathione S-transferase) fusion protein contg. the ligand binding domain of human PPAR?1 (KD = 70 nM). Using this ligand, the authors characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition expts., rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, resp.). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPAR?1 were comparable with those detd. in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an ?-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPAR?1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPAR? and that the pharmacol. of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

Aquaporins in adipocytes:insights into the pathophysiology of human obesity

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

PPARβ/δ ameliorates fructose-induced insulin resistance in adipocytes by preventing Nrf2 activation

We studied whether PPARβ/δ deficiency modifies the effects of high fructose intake (30% fructose in drinking water) on glucose tolerance and adipose tissue dysfunction, focusing on the CD36-dependent pathway that enhances adipose tissue inflammation and impairs insulin signaling. Fructose intake for 8weeks significantly increased body and liver weight, and hepatic triglyceride accumulation in PPARβ/δ-deficient mice but not in wild-type mice. Feeding PPARβ/δ-deficient mice with fructose exacerbated glucose intolerance and led to macrophage infiltration, inflammation, enhanced mRNA and protein levels of CD36, and activation of the JNK pathway in white adipose tissue compared to those of water-fed PPARβ/δ-deficient mice. Cultured adipocytes exposed to fructose also exhibited increased CD36 protein levels and this increase was prevented by the PPARβ/δ activator GW501516. Interestingly, the levels of the nuclear factor E2-related factor 2 (Nrf2), a transcription factor reported to up-regulate Cd36 expression and to impair insulin signaling, were increased in fructose-exposed adipocytes whereas co-incubation with GW501516 abolished this increase. In agreement with Nrf2 playing a role in the fructose-induced CD36 protein level increases, the Nrf2 inhibitor trigonelline prevented the increase and the reduction in insulin-stimulated AKT phosphorylation caused by fructose in adipocytes. Protein levels of the well-known Nrf2 target gene NAD(P)H: quinone oxidoreductase 1 (Nqo1) were increased in water-fed PPARβ/δ-null mice, suggesting that PPARβ/δ deficiency increases Nrf2 activity; and this increase was exacerbated in fructose-fed PPARβ/δ-deficient mice. These findings indicate that the combination of high fructose intake and PPARβ/δ deficiency increases CD36 protein levels via Nrf2, a process that promotes chronic inflammation and insulin resistance in adipose tissue.

Regulation of lipid metabolism in adipocytes and hepatocytes by hexarelin through scavenger receptor CD36

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides.

Differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis in brown adipocytes

Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-¹³C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-¹³C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine’s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin).

Combined transcriptomic-(1)H NMR metabonomic study reveals yhat monoethylhexyl phthalate stimulates adipogenesis and glyceroneogenesis in human adipocytes

Adipose tissue is a major storage site for lipophilic environmental contaminants. The environmental metabolic disruptor hypothesis postulates that some pollutants can promote obesity or metabolic disorders by activating nuclear receptors involved in the control of energetic homeostasis. In this context, monoethylhexyl phthalate (MEHP) is of particular concern since it was shown to activate the peroxisome proliferator-activated receptor γ (PPARγ) in 3T3-L1 murine preadipocytes. In the present work, we used an untargeted, combined transcriptomic-(1)H NMR-based metabonomic approach to describe the overall effect of MEHP on primary cultures of human subcutaneous adipocytes differentiated in vitro. MEHP stimulated rapidly and selectively the expression of genes involved in glyceroneogenesis, enhanced the expression of the cytosolic phosphoenolpyruvate carboxykinase, and reduced fatty acid release. These results demonstrate that MEHP increased glyceroneogenesis and fatty acid reesterification in human adipocytes. A longer treatment with MEHP induced the expression of genes involved in triglycerides uptake, synthesis, and storage; decreased intracellular lactate, glutamine, and other amino acids; increased aspartate and NAD, and resulted in a global increase in triglycerides. Altogether, these results indicate that MEHP promoted the differentiation of human preadipocytes to adipocytes. These mechanisms might contribute to the suspected obesogenic effect of MEHP.

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes

The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-(14)C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression of Clock, Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin temporally and rhythmically synchronized their metabolic and hormonal function in a circadian fashion, mimicking what is observed in vivo in animals during the daily light-dark cycle. Therefore, this work helps to clarify the physiological relevance of the circadian pattern of melatonin secretion and its interactions with Ins, contributing to a better understanding of the adipocyte biology.

Triiodothyronine Increases mRNA and Protein Leptin Levels in Short Time in 3T3-L1 Adipocytes by PI3K Pathway Activation

The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of leptin mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. We examined the involvement of this pathway in mediating TH effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI=100 nM) T3 dose during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey's test was used at 5% significance. T3 increased leptin mRNA expression in P (2.26 ± 0.36, p< 0.001), SI (1.99 ±0.22, p< 0.01) compared to C group (1± 0.18). This increase was completely abrogated by LY294002 in P (1.31±0.05, p< 0.001) and SI (1.33±0.31, p< 0.05). Western blotting confirmed these results at protein level, indicating the PI3K pathway dependency. To examine whether leptin is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). In P, the presence of CHX maintained the levels mRNA leptin, but was completely abrogated in SI (1.14±0.09, p> 0.001). These results demonstrate that the activation of the PI3K signaling pathway has a role in TH-mediated direct and indirect leptin gene expression in 3T3-L1 adipocytes. © 2013 Oliveira et al.

Short-term effects of triiodothyronine on thyroid hormone receptor alpha by PI3K pathway in adipocytes, 3T3-L1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background: Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B-1 receptor knockout mice (B-1(-/-)) are leaner and exhibit improved insulin sensitivity. Methodology/Principal Findings: Here we show that kinin B-1 receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B-1 receptors. In these cells, treatment with the B-1 receptor agonist des-Arg(9)-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B-1(-/-) mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B-1 receptor was limited to cells of the adipose tissue (aP2-B-1/B-1(-/-)). Similarly to B-1(-/-) mice, aP2-B-1/B-1(-/-) mice were leaner than wild type controls. However, exclusive expression of the kinin B1 receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B-1(-/-) mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B-1 receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B-1/B-1(-/-) when compared to B-1(-/-) mice. When subjected to high fat diet, aP2-B-1/B-1(-/-) mice gained more weight than B-1(-/-) littermates, becoming as obese as the wild types. Conclusions/Significance: Thus, kinin B-1 receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.