Adipokine expression profile in adipocytes of different mouse models of obesity.

Adipose tissue produces and secretes multiple adipokines. Most studies on adipokine production/expression have been performed on whole adipose tissue. In addition, data concerning an overall of adipokine expression are scarce and can be heterogeneous depending on the obesity model studied. Our first aim was to compare the expression of adipokines involved in the interplay between obesity and insulin resistance in isolated adipocytes from different mouse models of obesity displaying different levels of weight gain and insulin sensitivity. The second aim was to determine perigonadal/subcutaneous ratio of each adipokine. Only resistin expression was decreased in obese mice without modifications in glucose and insulin blood levels. In addition to decreased levels of resistin, obesity models associated with hyperglycemia and hyperinsulinemia presented an increased expression of leptin and tumor necrosis factor-alpha (TNFalpha). Obese and diabetic mice were the only animals to exhibit high expression of plasminogen activator inhibitor type-1 and interleukin-6. All adipokines except TNFalpha were more heavily expressed in perigonadal than in subcutaneous adipocytes. Interestingly, fat-enriched diet and overweight on their own did not modify the distribution of adipokines between the two fat depots. However, severe obesity modified the distribution of proinflammatory adipokines. In conclusion, the level and number of adipokines with altered expression increased with obesity and hyperinsulinemia in mice. The physiopathological impact of depot-specific differences of adipokine expression in adipocytes remains to be clarified.

Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression.

AIMS/HYPOTHESIS: Autotaxin is a lysophospholipase D that is secreted by adipocytes and whose expression is substantially up-regulated in obese, diabetic db/db mice. The aim of the present study was to depict the physiopathological and cellular mechanisms involved in regulation of adipocyte autotaxin expression. METHODS: Autotaxin mRNAs were quantified in adipose tissue from db/db mice (obese and highly diabetic type 2), gold-thioglucose-treated (GTG) mice (highly obese and moderately diabetic type 2), high-fat diet-fed (HFD) mice (obese and moderately diabetic type 2), streptozotocin-treated mice (thin and diabetic type 1), and massively obese humans with glucose intolerance. RESULTS: When compared to non-obese controls, autotaxin expression in db/db mice was significantly increased, but not in GTG, HFD, or streptozotocin-treated mice. During db/db mice development, up-regulation of autotaxin occurred only 3 weeks after the emergence of hyperinsulinaemia, and simultaneously with the emergence of hyperglycaaemia. Adipocytes from db/db mice exhibited a stronger impairment of insulin-stimulated glucose uptake than non-obese and HFD-induced obese mice. Autotaxin expression was up-regulated by treatment with TNFalpha (insulin resistance-promoting cytokine), and down-regulated by rosiglitazone treatment (insulin-sensitising compound) in 3T3F442A adipocytes. Finally, adipose tissue autotaxin expression was significantly up-regulated in patients exhibiting both insulin resistance and impaired glucose tolerance. CONCLUSIONS/INTERPRETATION: The present work demonstrates the existence of a db/db-specific up-regulation of adipocyte autotaxin expression, which could be related to the severe type 2 diabetes phenotype and adipocyte insulin resistance, rather than excess adiposity in itself. It also showed that type 2 diabetes in humans is also associated with up-regulation of adipocyte autotaxin expression.

Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.

Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.

In vitro and in vivo impairment of alpha2-adrenergic receptor-dependent antilipolysis by fatty acids in human adipose tissue.

The aim of the present study was to study the influence of fatty acids on the adrenergic control of lipolysis both in vitro and in vivo. Human subcutaneous adipose tissue explants were cultured for 48 h in the presence of 100 microM bromopalmitate (BrPal), and lipolysis was measured in isolated adipocytes. In control conditions, beta-AR-dependent activation of lipolysis by epinephrine was almost undetectable, and could be fully restored by pharmacological blockade of alpha2-AR-dependent antilipolysis. After BrPal treatment, epinephrine became fully lipolytic and was no longer influenced by alpha2-AR-blockade. Radioligand binding analysis revealed that BrPal treatment led to a significant reduction in the coupling of alpha2-AR to G proteins. In parallel, a chronic and significant increase in plasma fatty acids resulting from a 4-day high-fat diet (HFD) was accompanied by an impairment of the amplifying effect of the alpha2-AR antagonist phentolamine on exercise-induced lipolysis (measured in the subcutaneous adipose tissue with the use of a microdialysis probe) normally observed after a low-fat diet. In conclusion, in vitro and in vivo studies showed that fatty acids impair alpha2-AR-dependent antilipolysis.

Lysophosphatidic acid synthesis and release.

Lysophosphatidic acid (LPA) is a bioactive phospholipid controlling numerous cellular responses through the activation of specific G-protein coupled transmembrane receptors. LPA is present in several biological fluids (serum, plasma, aqueous humor) and can be secreted by several cell types (platelets, fibroblasts, adipocytes, cancer cells). Whereas, multiple pathways of synthesis and degradation of LPA have been described, their relative contribution in extracellular secretion and biodisponibility is still a matter of debate. The first part of the present review is devoted to the description of the different enzymes involved in LPA synthesis (acyltransferases, phospholipases, kinases) and degradation (lysophospholipases, lipid-phosphatases), as well as to the molecules involved in LPA transport (albumin, fatty acid binding proteins, gelsolin, lipoproteins). In a second part, the different physio-pathological situations (aggregation, cancer, injuries) associated with LPA production, as well as the potential role played by LPA in genesis of certain diseases (cancer, obesity, arteriosclerosis) are listed and analyzed.

Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans.

Analysis of the alpha2C-adrenergic receptor gene promoter and its cell-type-specific activity.

As an initial approach to define the regulatory elements and transcriptional factors that account for cell-restricted expression of the alpha2c-adrenergic receptor (AR) gene, we isolated and characterized the receptor gene and identified regions of the gene conferring cell-specific expression. A 4300-nucleotide (nt) fragment of the 5'-flanking region of the rat alpha2c-AR gene was isolated from a genomic library. The genomic sequence contained the uninterrupted sequence of the 5'-untranslated region of a previously isolated alpha2c-AR cDNA clone indicating the lack of introns in the 5' gene segment. RNase protection assays and/or RNA blot analysis indicated the expression of alpha2c-AR mRNA in rat brain but not in kidney or liver, which is consistent with the major expression of this gene in neuronal tissue. The 5' gene segment was used to identify sites of transcriptional initiation and promoter activity by RNase protection assays and transient transfection of reporter gene constructs. With the use of RNA probes progressively upstream of the translational start site, RNase protection assays with rat brain total RNA indicated multiple sites of transcriptional initiation within a approximately 70-nt span (-660 to -730 nt 5' to the translational start codon). The zone of transcriptional initiation was part of a larger GC-rich area of the 5' gene segment that is a characteristic of genes initiating transcripts at multiple sites. The promoter activity of this zone of transcriptional initiation and the influence of gene segments 5' to this area were addressed using chloramphenicol acetyl transferase reporter gene constructs. Transient transfection of reporter gene constructs indicated that a 96-nt gene fragment (-699/-603 relative to the translational start codon) was sufficient to direct transcription in the neuroblastoma X glioma hybrid cell line NG108-15, a cell line expressing the endogenous alpha2c-AR. Promoter activity was not observed in constructs lacking the zone of transcriptional initiation. The promoter segment was inactive when introduced into the rat glioma cell line C6B4, the rat submandibular cell line RSMT-A5, and the rat pancreatic beta cell line RIN-5AH, all of which do not express the endogenous alpha2c-AR gene. Upon incubation with nuclear extracts, a 129-nt fragment encompassing the promoter exhibited a gel mobility shift pattern that was specific for cells expressing the receptor protein and involved a nuclear protein that recognized a Sp1 oligonucleotide. These data indicate that a 96-nt gene promoter segment of the alpha2c-AR gene functions in a cell-type-specific manner.

Alpha 2-adrenergic stimulation promotes preadipocyte proliferation. Involvement of mitogen-activated protein kinases.

Primary cultures of rat preadipocytes were shown to express alpha 2- and beta-adrenoreceptors when maintained in serum-deprived medium. alpha 2-Adrenoreceptor stimulation led to an increase in cell number, whereas beta-adrenoreceptor stimulation was without effect. On 3T3-F442A clones stably transfected with the human alpha 2-C10 gene and expressing a physiologically related number of alpha 2-adrenoreceptors to overexpression, the proliferative effect of alpha 2-adrenoreceptor agonist UK14304 was proportional to the level of alpha 2-adrenoreceptor expressed in individual clones and required alpha-2 adrenoreceptor stimulation. Analysis of the signaling pathway linked to alpha 2-adrenoreceptor activation showed that the mitogenic effect was adenylyl cyclase- and protein kinase C-independent. It was pertussis toxin-sensitive, implying the involvement of pertussis toxin-sensitive G proteins. The activation of the mitogen-activated protein kinase pathway was increased after alpha 2-adrenoreceptor stimulation in both 3T3-F442 clones and rat preadipocytes and inhibited by pertussis toxin treatment. So, catecholamines are involved in the control of white preadipocyte proliferation through the alpha 2-adrenoreceptor activation, linked to the mitogen-activated protein kinase pathway.

In vivo upregulation of adipocyte alpha 2-adrenoceptors by androgens is consequence of direct action on fat cells.

Adipose precursor cells from male hamsters were exposed to testosterone in primary culture. The effect of the sex steroid on alpha 2-adrenoceptor (AR) expression was studied. Testosterone enhanced alpha 2-AR expression during adipose conversion to an identical extent to that determined in vivo without modification of the differentiation state as estimated by glycerol-3-phosphate dehydrogenase activity. The expression of preadipocyte alha 2-ARs from female hamster was also submitted to the testosterone control. The upregulating effect was androgen specific, since only testosterone and dihydrotestosterone were efficient in inducing an increase of alpha 2-AR density, whereas estradiol, dexamethasone, progesterone, and androstenedione were without any action. To conclude, androgens act directly on fat cells by upregulating alpha 2-AR expression. The effects are not mediated by aromatization into estrogens and probably involve androgen receptor interactions.

Androgenic regulation of adipocyte alpha 2-adrenoceptor expression in male and female Syrian hamsters: proposed transcriptional mechanism.

Adaptation of male hamsters to short daily (SD) photoperiod induced a reduction of the adipocyte alpha 2-adrenoceptor (alpha 2-AR) expression which was related to a sexual involution and could be reversed by testosterone administration. In the present paper, the possible mechanisms of such a physiological regulation are explored. The effect of testosterone on the adipocyte alpha 2-AR was rapid, dose-dependent, occurred at the physiological plasma concentrations of androgen, and was mimicked by dihydrotestosterone, but not by 17 beta-estradiol, progesterone, hydrocortisol, insulin, or T3. Adaptation of female hamsters to SD photoperiod also induced a sexual involution, but no modification of the adipocyte alpha 2-AR number was observed. Administration of testosterone induced a large up-regulation of the adipocyte alpha 2-AR. Testosterone was also able to up-regulate the adipocyte alpha 2-AR in male hamsters adapted to long day photoperiod whatever their age (6-, 12-, and 25-week old). Adaptation to SD photoperiod did not modify the adipocyte adenylyl cyclase activity (basal, forskolin-stimulated, GppNHp-inhibited). Conversely, UK14304-mediated inhibition of the adenylyl cyclase was suppressed in SD photoperiod and recovered after testosterone treatment. Administration of testosterone in young male hamsters adapted to long day photoperiod induced an increase in the amount of the alpha 2-AR messenger RNA, which coincided with the increase in the alpha 2-AR maximal number. The existence of a specific regulation of the adipocyte alpha 2-AR by the androgens, probably via a transcriptional mechanism, can be hypothesized. This regulation, which occurs in both male and female hamsters, appears to be physiologically relevant.

Late expression of alpha 2-adrenergic-mediated antilipolysis during differentiation of hamster preadipocytes.

In order to study the ontogenesis of the beta- and alpha 2-adrenergic control of lipolysis during the adipose conversion process, a model based on preadipocytes isolated from the stromal-vascular fraction of hamster adipose tissue was developed. When cultured in an ITT (insulin, transferrin, triiodothyronine) medium supplemented with 2% fetal calf serum, adipose precursors differentiated into adipose-like cells. On 8-day-post-confluent differentiating preadipocytes, the rank order of potency of activation of lipolysis by various beta-adrenergic agonists (BRL37344 greater than norepinephrine = isoproterenol greater than epinephrine greater than fenoterol) was equivalent to that determined in mature adipocytes isolated from adult hamster adipose tissue. On 8-day-post-confluent differentiating preadipocytes, phenylisopropyladenosine (A1-adenosine agonist) and prostaglandin E1 evoked a strong antilipolytic response whereas that evoked by UK 14304 and clonidine (alpha 2-adrenergic agonists) remained undetectable at this step of differentiation. The activity of UK 14304 and clonidine only appeared on 20- to 25-day-post-confluent differentiating preadipocytes. They induced dose-dependent antilipolysis with a maximal effect reaching 80-85% inhibition of adenosine deaminase-stimulated lipolysis. Their action was blocked by increased concentrations of different alpha 2-adrenergic antagonists with the following order of potency, RX 821002 greater than phentolamine much greater than yohimbine. This order of potency was similar to that determined on mature adipocytes isolated from adult hamsters. Both the density of the alpha 2-adrenoceptors, identified with the selective alpha 2-adrenergic radioligand [3H]RX-821002 (19 +/- 1 vs. 30 +/- 1 fmol/mg protein: P less than 0.01) and the amount of Gi proteins identified by pertussis toxin-catalyzed ADP-ribosylation (31 +/- 4 vs. 43 +/- 4% of the amount defined in mature fat cells from adult hamsters: P less than 0.05) were significantly increased between 8 days and 20-25 days after confluence, explaining the late emergence of the alpha 2-adrenergic control of lipolysis during preadipocyte differentiation. In conclusion, the late emergence of the alpha 2-adrenergic control of lipolysis, which is also supported by previous data obtained in vivo that demonstrated the age and/or the fat cell size dependence of alpha 2-adrenoreceptor expression in mature adipocytes, allows the alpha 2-adrenoceptor to be considered as a marker of adipocyte hypertrophy.

Photoperiodic control of adipocyte alpha 2-adrenoceptors in Syrian hamsters: role of testosterone.

The impact of photoperiodic manipulations and testosterone treatments on the adipocyte alpha 2-adrenergic (alpha 2-AR), beta-adrenergic (beta-AR), and A1-adenosine (A1-R) responsiveness, was explored in male Syrian hamsters (Mesocricetus auratus). Moreover, binding studies were performed with appropriate alpha 2-AR, beta-AR, and A1-R radioligands to study receptor changes. Animals were kept for 12 weeks in long day photoperiod (LD: 16 h light (L)-8 h dark (D)), in short day photoperiod (SD: 6L-18D), or in short photoperiod with testosterone treatments (1 mg/animal/day sc) 10 days before sacrifice (SD+T). The antilipolytic effect of the full alpha 2-AR agonist UK14304 and the specific binding of the alpha 2-AR radioligands [3H] RX821002 (antagonist) and [3H]UK14304 were significantly reduced in SD hamsters compared with LD hamsters. The alpha 2-site number and alpha 2-AR responsiveness were completely restored in SD+T hamsters. Whatever the experimental conditions the adipocyte beta-AR receptivity (lipolytic response of isoproterenol and [125I]cyanopindolol binding), and the A1-R receptivity (antilipolytic response initiated by (-)phenylisopropyladenosine and [3H]dipropyl-8-cyclopentylxanthine and [3H]phenylisopropyladenosine binding) remained unchanged. Moreover, the kidney and brain alpha 2-AR densities identified with [3H]RX821002 were not significantly different in LD, SD or SD+T hamsters. These results were obtained without any modification of animal weight, white adipose tissue weight, or white fat cell size. We conclude that, in the Syrian hamster, the expression of the adipocyte alpha 2-AR is under the control of the photoperiod by a testosterone-dependent mechanism probably mediated through the hypothalamic-pituitary axis, without any alteration of the animal fat stores.

Selective reduction of alpha 2-adrenergic responsiveness in hamster adipose tissue during prolonged starvation.

The influence of fasting on the dual adrenergic control of adipose tissue lipolysis was investigated in hamsters because in this species the adipocytes exhibit both beta-stimulatory and alpha 2-inhibitory adrenergic responses. In adipocytes from fed animals, the number of alpha 2-receptors (identified with [3H]clonidine and [3H]RX 821002) was greater than that of beta-receptors. As in humans, the alpha 2-adrenoceptor number was greater in adipocyte membranes from subcutaneous (inguinal and popliteal) than from internal (perirenal and epididymal) adipose tissues. Despite this difference in alpha 2-adrenoceptor number, the antilipolytic responses to the alpha 2-agonists clonidine and UK 14304 were similar in the two tissues. Food deprivation for a period of 1-6 days induced a net depletion of both adipose tissues. In 6-day starved animals the number of adipocyte alpha 2-adrenoceptors and the maximal antilipolytic effect of UK 14304 were less than 50% of those in fed controls. In contrast, the antilipolytic responses to phenylisopropyladenosine or prostaglandin E1 remained unchanged. Starvation induced a decrease in alpha 2-adrenoceptor number and an increase in beta-adrenergic sensitivity that were greater in adipocytes from subcutaneous than from internal fad pads. The data suggest that the adipocyte beta- and alpha 2-adrenoceptors are independently regulated during starvation. In the adipocyte, the alpha 2-antilipolytic responses and the alpha 2-adrenoceptor levels are dependent on the extent of the adipose mass; they are particularly reduced in emaciated hamsters.

Lipolytic action of a new alpha-2 adrenergic antagonist of the piperazinopyrimidine family: RP 55462.

Recent investigations have demonstrated the in vitro lipid mobilizing effects induced by alpha-2 adrenergic antagonist administration and have focused attention on the putative therapeutic interest of such compounds in the treatment of obesity as adjuvants in caloric restriction programs. We studied the impact of RP 55462 [6-chloro-4-(isopropylamino)-5-(methyl)-2, piperazinopyrimidine], a new alpha-2 adrenergic antagonist compound of the piperazinopyrimidine family, on fat cell function. The alpha-2-blocking properties of this agent, which had been defined initially on the brain were confirmed on adipocytes. RP 55462 competed with [3H]yohimbine binding sites on human fat cell membranes and inhibited the antilipolytic action of alpha-2-agonist compounds (UK 14304, clonidine and epinephrine) in human and hamster fat cells. It was also noticed that RP 55462 alone was able to activate lipolysis in isolated fat cells from various species (man, rat, hamster and dog). Moreover, the lipolytic response induced by isoproterenol or synacthene was largely amplified in the presence of RP 55462 in rat fat cells which are the least alpha-2 adrenergic responsive tested. RP-55462-dependent stimulation of lipolysis was not affected by the presence of other alpha-2 adrenergic antagonists (idazoxan, yohimbine or phentolamine). Intravenous administration of RP 55462 in alert dogs promoted an increment in plasma nonesterified acid concentrations reflecting its lipid mobilizing action. In summary this study focuses attention on a new alpha-2-antagonist compound which exhibits an in vivo lipid mobilizing action which could be attributable to its alpha-2 adrenergic antagonist properties. Inasmuch as the lipolytic activity of RP 55462, revealed in in vitro studies, seems to be independent from its alpha-2 adrenolytic properties; further studies are required to define the mechanism of such a lipolytic effect as well as its possible involvement in in vivo conditions.

Mechanism of lipolytic action of a new alpha-2 adrenergic antagonist of the piperazinopyrimidine family: RP 55462.

The in vivo lipid mobilizing effect of alpha-2 adrenergic antagonist has been demonstrated previously. This has attracted attention to the putative interest of such compounds in a lipid-mobilizing strategy. RP 55462 [6-chloro-4-(isopropylamino)-5-(methyl)-2 piperazinopyrimidine], a piperazinopyrimidine derivative, has already been shown to exert alpha-2 adrenergic antagonist actions on fat cell function in vitro. Moreover, RP 55462 exhibits a direct in vitro lipolytic action which is independent of its alpha-2-blocking potency. When administered i.v. RP 55462 is also able to induce an increment in plasma nonesterified levels in dogs. The mechanism of action of RP 55462 was studied and the nature of its lipomobilizing effect was explored. RP 55642-dependent lipolysis was not affected by beta adrenergic blockers on rat fat cells and RP 55462 had no direct effect on adenylylcyclase activity on fat cell membranes. Moreover, RP 55462 did not compete with [3H]phenyl isopropyl adenosine binding (A1-adenosine receptor agonist) on fat cell membranes. In fact, RP 55462 inhibited, in a dose-dependent manner, the cyclic AMP (cAMP)-dependent phosphodiesterase (PDE) activity in rat adipose tissue. Several derivatives with the piperazinopyrimidine structure also inhibited cAMP-dependent PDE activity and exerted lipolytic effects. A short structure-activity study was performed with various derivatives. In dogs, by contrast with yohimbine, the in vivo lipid mobilizing effect of RP 55462 was not abolished by pretreatment with propranolol, and lasted longer. It is concluded that the in vivo lipolytic activity of RP 55462 is connected with its ability to inhibit cAMP-dependent PDE activity; a property of several piperazinopyrimidine derivatives. The lipid mobilizing effect induced in vivo by RP 55462 results from a combination of its alpha-2 adrenergic antagonist properties and its direct lipolytic action mediated by cAMP-dependent PDE inhibiting effects.