Influence of high-fat diet on amine oxidase activity in white adipose tissue of mice prone or resistant to diet-induced obesity.

Decreased monoamine oxidase (MAO) activity has been observed in adipose tissue of obese patients. Since substrates of MAO and semicarbazide-sensitive amine oxidase (SSAO) can modify adipocyte metabolism, this work investigates whether changes in amine oxidase activity may occur during white adipose tissue (WAT) development. We evaluated MAO and SSAO activities in WAT of high-fat diet (HFD) and low-fat diet fed mice. To distinguish the effect of HFD on its own from the effect of fat mass enlargement, obesity-prone transgenic line of the FVBn strain lacking beta3-adrenergic receptors (AR) but expressing human beta3-AR and alpha2-AR (mbeta3-/-, hbeta3+/+, halpha2+/-) was compared to its obesity-resistant control (mbeta3-/-, hbeta3+/+). As already reported, the former mice became obese while the latter resisted to HFD. No significant change in SSAO or MAO activity was found in WAT of both strains after HFD when expressing oxidase activity per milligram of protein. However, when considering the overall capacity of the fat depots to oxidize tyramine or benzylamine, there was an increase in MAO and SSAO activity only in the enlarged WAT of HFD-induced obese mice. Therefore, the comparison of these models allowed to demonstrate that the higher amine oxidase capacity hold in enlarged fat stores of obese mice is more likely the consequence of increased fat cell number rather than the result of an increased expression of MAO or SSAO in the adipocyte.

Influence of high-fat diet on amine oxidase activity in white adipose tissue of mice prone or resistant to diet-induced obesity

Decreased monoamine oxidase (MAO) activity has been observed in adipose tissue of obese patients. Since substrates of MAO and semicarbazide-sensitive amine oxidase (SSAO) can modify adipocyte metabolism, this work investigates whether changes in amine oxidase activity may occur during white adipose tissue (WAT) development. We evaluated MAO and SSAO activities in WAT of high-fat diet (HFD) and low-fat diet fed mice. To distinguish the effect of HFD on its own from the effect of fat mass enlargement, obesity-prone transgenic line of the FVBn strain lacking b3-adrenergic receptors (AR) but expressing human b3-AR and a2-AR (mb3-/-, hb3+/+, ha2+/-) was compared to its obesity-resistant control (mb3-/-, hb3+/+). As already reported, the former mice became obese while the latter resisted to HFD. No significant change in SSAO or MAO activity was found in WAT of both strains after HFD when expressing oxidase activity per milligram of protein. However, when considering the overall capacity of the fat depots to oxidize tyramine or benzylamine, there was an increase in MAO and SSAO activity only in the enlarged WAT of HFD-induced obese mice. Therefore, the comparison of these models allowed to demonstrate that the higher amine oxidase capacity hold in enlarged fat stores of obese mice is more likely the consequence of increased fat cell number rather than the result of an increased expression of MAO or SSAO in the adipocyte

En trabajos previos se describe que la actividad monoamino oxidasa (MAO) en el tejido adiposo disminuye en pacientes obesos. Dado que los sustratos de la MAO y de la amino oxidasa sensible a semicarbazida (SSAO) pueden modificar el metabolismo de los adipocitos, se investiga en este trabajo si se producen cambios en la actividad amino oxidasa durante el desarrollo del tejido adiposo blanco. Para ello, se evalúa el efecto de la dieta rica en grasa (HFD) respecto de la dieta control sobre la actividad MAO y SSAO en tejido adiposo de ratones transgénicos que no expresan sus propios receptores adrenérgicos b3 y que son de dos líneas diferentes: los que expresan los receptores adrenérgicos humanos b3 Y a2 (mb3 -/-, hb3+/+, ha2+/-) y que son no susceptibles a la obesidad y los que sólo expresan los b3 humanos (mb3 -/-, hb3+/+) y que son resistentes a la obesidad. Los resultados no muestran cambios significativos por efecto de la dieta en ninguno de los dos grupos de ratones sobre la actividad MAO y SSAO cuando se expresa referida a mg de proteína. Sin embargo, cuando se considera la capacidad total de los depósitos grasos para oxidar tiramina o benzilamina, so observa un aumento significativo en ambas actividades MAO y SSAO sólo en el incrementado tejido adiposo blanco de los ratones obesos por efecto de la dieta HFD. Por tanto, estos datos indican que la mayor actividad amino oxidasa de los depósitos grasos de los ratones obesos es probablemente debida al aumento del número de células adiposas, mas que a un incremento de la expresión de MAO y SSAO en los adipocitos

Dual action of octopamine on glucose transport into adipocytes: inhibition via beta3-adrenoceptor activation and stimulation via oxidation by amine oxidases.

Octopamine, which is closely related to norepinephrine, acts as a neurotransmitter in invertebrates and is a trace amine with undefined properties in vertebrates. The octopaminergic receptors identified in insects are targets of various pesticides but are absent in vertebrates. We have established that octopamine stimulates fat cell lipolysis in mammals via activation of beta3-adrenoceptors (ARs), whereas this amine has been described elsewhere as an alpha2-AR agonist and as a substrate for monoamine oxidase (MAO) or semicarbazide-sensitive amine oxidase (SSAO). Because we have recently reported that amine oxidase substrates promote glucose transport in rat and human adipocytes, the in vitro octopamine effects on lipolysis and glucose uptake were reassessed by using adipocytes from beta3-AR-deficient mice. The lipolytic effect and the counter-regulation of insulin action on glucose transport provoked by 0.1 to 1 mM octopamine or by 1 microM beta3-AR agonists found in control animals disappeared in adipocytes from beta3-AR-deficient mice. This revealed an insulin-like effect of octopamine on glucose uptake, which was dependent on its oxidation by MAO or SSAO, as was the case for tyramine and benzylamine, devoid of beta3-adrenergic agonism. Similarly, octopamine promoted glucose transport in human adipocytes and exhibited a weaker lipolytic stimulation than in rodent adipocytes. These findings indicate that, besides its lipolytic activity, octopamine exerts, at millimolar dose, dual effect on glucose transport in adipocytes: counteracting insulin action via beta3-AR activation and stimulating basal transport via its oxidation by MAO or SSAO.

Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis.

Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these alpha/beta hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.

Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells.

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes.

Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01-1 microM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and/or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline) and PI-3K-dependent (in the presence of 100 nM isoprenaline) anti-lipolytic pathways, both of which were found to be independent of phosphodiesterase type 3B (PDE3B). pV (0.01 mM), like insulin, activated PI-3K- and PDE3B-dependent pathways. However, the anti-lipolytic pathway of 0.1 mM pV did not seem to require insulin receptor substrate-1-associated PI-3K and was found to be partly independent of PDE3B. Vanadate and pV (only at 0.01 mM), like insulin, decreased the isoprenaline-induced activation of cAMP-dependent protein kinase. Overall, these results underline the complexity and the diversity in the mechanisms that regulate lipolysis.

Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate.

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.

Adrenergic receptors and fat cells: differential recruitment by physiological amines and homologous regulation.

The control of fat cell lipolysis by the catecholamines involves at least four different adrenoceptor subtypes; three beta (beta 1-, beta 2-, and beta 3-ARs) and one alpha 2-adrenoceptor (alpha 2-AR). The physiological importance of the beta- and alpha 2A-ARs varies according to the species, the sex, the age, the anatomical location of fat deposits and the degree of obesity in humans and animals. The physiological amines operate through differential recruitment of these sites on the basis of their relative affinities. This point has been assessed by in vitro studies and has partly been confirmed in in vivo experiments using selected alpha/beta-AR antagonists and in situ microdialysis. The affinity of the beta 3-AR for catecholamines is less than that of the classical beta 1- and beta 2-ARs in the various species investigated. Conversely, it is the alpha 2-AR which exhibit the highest affinity for the physiological amines in all fat cells. The relative order of affinity of the various fat cell ARs for the physiological amines defined in binding studies and in vitro assays is alpha 2 > beta 1 > or = beta 2 > beta 3 for norepinephrine and alpha 2 > beta 2 > beta 1 > beta 3 for epinephrine. When considering differential beta-AR recruitment by catecholamines, it is the beta 1-AR which is always activated at the lowest norepinephrine levels, whatever the species, while the activation of the beta 3-AR requires higher norepinephrine levels. In addition to the differential recruitment, differential regulation by hormones could also occur for each fat cell AR subtype. The alpha 2-and beta 3-ARs are less prone to desensitization and down-regulation by comparison with the beta 1- and beta 2-AR.

Human adipocytes express alpha 2-adrenergic receptor of the alpha 2A-subtype only: pharmacological and genetic evidence.

In the present study we have reinvestigated the subtype of alpha 2-adrenoceptors expressed in human adipocytes (from subcutaneous and internal fat deposits) by means of radioligand binding using subtype-selective antagonists, and RNase mapping using a set of specific probes prepared from human alpha 2-adrenoceptors genes (alpha 2C2, alpha 2C4 and alpha 2C10). Comparison of the pharmacological properties of the human adipocyte alpha 2-adrenoceptors with those of the different human adrenoceptors expressed in COS-7 cells demonstrated that: i) human adipocyte alpha 2-adrenoceptors displays a KD for [3H]RX821002 and [3H]MK912 identical to that found in COS-7 cells transfected with the alpha 2C10 gene; ii) yohimbine and oxymetazoline is 1,000-fold more potent than prazosin to inhibit [3H]antagonist binding. RNase protection assays on cellular RNA prepared from the three fat deposits showed the presence of substantial amounts of alpha 2C10 transcripts: in contrast, mRNAs from alpha 2C2 and alpha 2C4 genes were undetectable. Altogether these results definitively establish that human adipocytes express only one alpha 2-adrenoceptor which is of the alpha 2A-subtype and encoded by the alpha 2C10 gene.

Antilipolytic effects of alpha 2-adrenergic agonists, neuropeptide Y, adenosine, and PGE1 in mammal adipocytes.

Lipolysis is stimulated by different hormones, depending on the species, but is also regulated by antilipolytic modulators, such as catecholamines (alpha 2-agonists), neuropeptide Y (NPY), adenosine, and prostaglandin E1 (PGE1), for which species-specific variations are poorly described. Comparison of the efficiency of these antilipolytic systems showed that PGE1 or phenylisopropyladenosine was able to totally inhibit lipolysis activation in nine mammalian species. However, the antilipolytic responses to clonidine or UK-14304 were fully developed in hamster and rabbit but blunted in the adipocytes of jerboa, rat, guinea pig, garden dormouse, and dormouse. A powerful antilipolytic effect of NPY was found only in the garden dormouse. Only human and dog adipocytes exhibited antilipolytic responses to alpha 2-adrenergic and NPY stimulation. These observations were explained by differences in alpha 2-adrenergic and NPY/peptide YY receptor number. Thus, inhibitory regulation of lipolysis in white adipocytes seems to be composed of two systems: a constitutive one that is related to paracrine mediators (adenosine, prostaglandins) and a regulatory one including neuroendocrine messengers such as catecholamines and NPY.

Adrenergic lipolysis in guinea pig is not a beta 3-adrenergic response: comparison with human adipocytes.

beta 3-Adrenoceptor agonists are potent lipolytic activators in rats, but they are only weak stimulators in human adipocytes, indicating interspecies differences in the adrenergic regulation of lipid mobilization. Like human but not rat adipocytes, guinea pig fat cells were poorly responsive to the beta 3-agonists BRL-37344, CGP-12177, SR-58611, and ICI-215001, acid metabolite of ICI-D7114. In guinea pigs, the beta 1-agonist dobutamine was more lipolytic than the beta 2-agonist procaterol. Anatomic location of fat deposits was without major influence on the beta-adrenergic responsiveness. Weak responses to beta 3-agonists were found whatever the sex or the age (from 2 days to 16 mo) of the animals. Even in the interscapular brown adipose tissue, which is well known in rats for its beta 3-adrenergic responsiveness, a blunted response to BRL-37344 was observed. The alpha 2-adrenergic antilipolytic effect and receptor number were smaller in guinea pig than in human adipocytes, but the beta-adrenergic receptor number was similar in the two species. Thus guinea pig adipocytes resemble human fat cells when their weak beta 3-adrenergic responsiveness is considered.

Distribution of PYY receptors in human fat cells: an antilipolytic system alongside the alpha 2-adrenergic system.

The antilipolytic effect of peptide YY (PYY) and neuropeptide Y has recently been shown in human adipocytes. PYY receptors were investigated in three human adipose deposits. A greater number of 125I-labeled PYY binding sites was found in femoral adipocyte membranes (maximal binding = 40 +/- 4 fmol/mg protein; dissociation constant = 0.3 +/- 0.1 nM) when compared with mammary and pericolonic adipose tissue. PYY receptors, like alpha 2-adrenergic sites, were largely expressed in femoral fat cells. Such a distribution was not specific either to inhibitory or to stimulating adenylyl cyclase systems since adenosine A1 and beta-adrenergic receptors were more numerous in pericolonic adipocytes. On isolated adipocytes, PYY (10(-7) M) inhibited lipolysis by 58 +/- 2% in femoral and 14 +/- 4% in pericolonic fat cells; epinephrine had the following similar response: 62 +/- 5 and 26 +/- 8%, respectively. A close relationship between the number of alpha 2-sites and PYY sites and the antilipolytic effects initiated by PYY and an alpha 2-agonist was observed. No significant differences were noted in the amount of Gi proteins in femoral and pericolonic adipocyte membranes.

Identification and functional studies of a specific peptide YY-preferring receptor in dog adipocytes.

Specific binding sites for peptide YY (PYY) and neuropeptide Y (NPY) as well as functional responses were identified in dog adipocytes. Studies were carried out using the radioligand [125I-Tyr1]monoiodo-PYY on crude adipocyte membranes. [125I]PYY bound to dog adipocyte membranes with a high affinity (156 +/- 24 pM) and binding capacity of 314 +/- 48 fmol/mg protein. Competition studies revealed a higher affinity of the binding sites for PYY than NPY (inhibition constants were 118 +/- 17 pM and 300 +/- 53 pM, respectively, P < or = 0.001). NPY analogs displaced [125I]PYY specific binding with the following order of potency: NPY-(13-36) > NPY-(18-36) > NPY-(22-36) >> [Leu31-Pro34]NPY. Neither adrenergic nor adenosine agents (activating or inhibiting other antilipolytic systems) interacted with [125I]PYY binding sites. So [125I]PYY binding was specific, saturable, and reversible. Lipolysis experiments performed with PYY, NPY, and NPY analogs confirm the relative order of potency found in competition experiments. The data agree with the definition of PYY-preferring receptor which resembles a Y2 receptor subtype since NPY-(13-36), a specific Y2 receptor agonist, inhibited binding and lipolysis in a similar way to PYY, whereas [Leu31-Pro34]NPY did not. No difference was observed in the antilipolytic response between IC50 values measured on omental, perirenal, and subcutaneous fat deposits. Moreover, PYY and NPY (10(-6) M) significantly attenuated forskolin-stimulated cAMP levels, involving inhibition of adenylyl cyclase as a transmembrane signaling mechanism. Cross-linking of bound [125I]PYY to membranes indicated that the mol wt of the receptor was 62K. The relative importance of such a receptor on fat cells alongside another powerful antilipolytic receptor--the alpha 2-adrenoceptor--is discussed.

Fate of exogenous and newly synthesized cholesterol in intestinal cell lines.

1. The current study was undertaken to test the existence of functionally distinct intracellular pools of cholesterol depending on the origin: neosynthesis or exogenous. 2. This was performed on two subpopulations, either differentiated or undifferentiated, of the HT29 cell line. 3. A parallel study was also carried out on Caco-2 cells. 4. First we checked the ability of differentiated HT29 cells to secrete lipids into the medium and found that lipid production was efficient but less so than in Caco-2 cells. 5. In contrast, undifferentiated HT29 cells were unable to secrete lipids into the medium. 6. Then we studied the fate of [14C]cholesterol incorporated into micellar preparations and of [14C]mevalonate in the different models. 7. The data obtained with labelled exogenous cholesterol show that it enters the membrane cholesterol pool as well as, for the differentiated models, the cholesteryl ester pool. 8. Similarly, labelled newly synthesized cholesterol could be used for membrane formation as well as for incorporation into cholesteryl esters. 9. Thus, in HT29 subpopulations as well as in Caco-2 cells, the results suggest the existence of a common pool of cholesterol whatever its origin.

Acyl-CoA: cholesterol acyltransferase in HT 29 cell subpopulations. Defect of activity in the undifferentiated cells.

The ACAT activity was studied on different subpopulations deriving from HT29 cells, a human colon carcinoma cell line. Grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G + cells). From this heterogeneous population, differentiated cells were selected by glucose deprivation and grown either on medium without glucose (G - cells) or in standard medium containing 25 mM glucose (G-Rev cells). The G- and G-Rev cells have the features of differentiated small intestine cells. The two types of differentiated cells (G- and G-Rev) exhibited similar ACAT activities and the kinetic characteristics of the enzyme were also similar. A time-course study showed increasing activity during the exponential phase and a decrease just after confluency. It was possible to stimulate the enzyme by micellar or lipoprotein cholesterol. In contrast, the ACAT activity was hardly detectable in undifferentiated G + cells. In addition, all the experimental conditions known to stimulate ACAT activity, and confirmed in the differentiated HT29 cells, were inefficient in the undifferentiated G + cells. Therefore, the different models derived from HT29 cells provide the opportunity to study cholesterol esterification as well as the consequences of its aberrances in intestinal cells.