Christian Carpéné

TNFα up-regulates apelin expression in human and mouse adipose tissue

We have recently identified apelin as a novel adipokine up‐regulated by insulin and obesity. Since obesity and insulin resistance are associated with chronically elevated levels of both insulin and TNFα, the present study was performed to investigate a putative regulation of apelin expression in adipocytes by TNFα. Herein, we report a tight correlation between apelin and TNFα expression in adipose tissue of lean and obese humans. Apelin regulation by TNFα was further studied in cultured explants of human adipose tissue. The endogenous expression of TNFα in adipocytes isolated from the explants was accompanied by a 6–9 h subsequent increase of apelin expression in adipocytes. This increase was reversed by inhibiting TNFα expression with 100 µM isobutylmethylxanthine. In different mouse models of obesity, expression of both TNFα and apelin was also significantly increased in adipocytes of obese mice. Furthermore, short‐term exposure to an i.p. injection of TNFα in C57Bl6/J mice induced an increase of apelin expression in adipose tissue as well as apelin plasma levels. Finally, a direct positive effect of TNFα has been shown in differentiated 3T3F442A adipocytes on apelin expression and secretion. The signaling pathways of TNFα for the induction of apelin were dependent of PI3‐kinase, c‐Jun NH2‐terminal kinase (JNK), and MAPK but not PKC activation. All together, these findings suggest that apelin might be a candidate to better understand potential links between obesity and associated disorders such as inflammation and insulin resistance.—Daviaud, D., Boucher, J., Gesta, S., Dray, C., Guigne, C., Quil‐liot, D., Ayav, A., Ziegler, O., Carpene, C., Saulnier‐Blache, J.‐S., Valet, P., Castan‐Laurell, I. TNFα up‐regulates apelin expression in human and mouse adipose tissue. FASEB J. 20, E796–E802 (2006)

Lipolysis is altered in MHC class I zinc-α2-glycoprotein deficient mice

Non‐conventional major histocompatibility complex class I molecules are involved in a variety of physiological functions, most at the periphery of the immune system per se. Zinc‐α2‐glycoprotein (ZAG), the sole soluble member of this superfamily has been implicated in cachexia, a poorly understood yet life‐threatening, severe wasting syndrome. To further ascertain the role of ZAG in lipid metabolism and perhaps the immune system, we inactivated both ZAG alleles by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild‐type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including β3‐adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.

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

Aims/hypothesisAutotaxin 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.MethodsAutotaxin 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.ResultsWhen 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 TNFα (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/interpretationThe 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.

Apelin stimulates glucose uptake but not lipolysis in human adipose tissue ex vivo

Apelin is a peptide present in different cell types and secreted by adipocytes in humans and rodents. Apelin exerts its effects through a G-protein-coupled receptor called APJ. During the past years, a role of apelin/APJ in energy metabolism has emerged. Apelin was shown to stimulate glucose uptake in skeletal muscle through an AMP-activated protein kinase (AMPK)-dependent pathway in mice. So far, no metabolic effects of apelin have been reported on human adipose tissue (AT). Thus, the effect of apelin on AMPK in AT was measured as well as AMPK-mediated effects such as inhibition of lipolysis and stimulation of glucose uptake. AMPK and acetyl-CoA carboxylase phosphorylation were measured by western blot to reflect the AMPK activity. Lipolysis and glucose uptake were measured, ex vivo, in response to apelin on isolated adipocytes and explants from AT of the subcutaneous region of healthy subjects (body mass index: 25.6 ± 0.8 kg/m(2), n = 30 in total). APJ mRNA and protein are present in human AT and isolated adipocytes. Apelin stimulated AMPK phosphorylation at Thr-172 in a dose-dependent manner in human AT, which was associated with increased glucose uptake since C compound (20u200a μM), an AMPK inhibitor, completely prevented apelin-induced glucose uptake. However, in isolated adipocytes or AT explants, apelin had no significant effect on basal and isoprenaline-stimulated lipolysis. Thus, these results reveal, for the first time, that apelin is able to act on human AT in order to stimulate AMPK and glucose uptake.

Tyramine Stimulates Glucose Uptake in Insulin-Sensitive Tissues in Vitro and in Vivo via Its Oxidation by Amine Oxidases

Tyramine and benzylamine have been described as stimulators of glucose transport in adipocytes. This effect is dependent on amine oxidation by monoamine oxidase (MAO) or semicarbazide-sensitive amine oxidase (SSAO) and on the subsequent hydrogen peroxide formation as already demonstrated by blockade with oxidase inhibitors or antioxidants and potentiation with vanadate. In this work, we extended these observations to skeletal muscle and cardiac myocytes using in vitro and in vivo approaches. Tissue distribution studies showed that substantial extrahepatic peripheral MAO activities exist in kidney and gut, but also in insulin-sensitive tissues: heart, adipose tissue, and skeletal muscles. SSAO activity is also widely distributed and present at a lower level than MAO, except in fat depots where both oxidases were equally involved in tyramine oxidation. When tested in vitro at millimolar doses, tyramine caused a large stimulation of glucose transport in rat adipocytes and in skeletal and cardiac muscles. In vivo administration of tyramine (4 mg/kg i.p.) lowered the hyperglycemic responses to a glucose challenge in control and in streptozotocin-treated rats. This positive effect on glucose disposal was obtained without vanadate and was abolished by SSAO and MAO inhibitors. Tyramine increased hexose uptake in vivo in insulin-sensitive tissues, whereas it induced only transient effects on plasma insulin or cardiovascular parameters. In conclusion, activation of the amine oxidases present in insulin-sensitive tissues induces insulin-like effects, readily detectable in vitro, and increasing peripheral glucose utilization in vivo.

High expression of monoamine oxidases in human white adipose tissue : Evidence for their involvement in noradrenaline clearance

The clearance of plasma adrenaline and noradrenaline by human adipose tissue suggests the expression of the catecholamine-degrading enzyme monoamine oxidases and of catecholamine transport systems in adipocytes. In the present study, we identified and characterized the monoamine oxidases and an extraneuronal noradrenaline transporter expressed in human adipocytes. Enzyme assays using the monoamine oxidase A/B substrate [14C]tyramine showed that abdominal and mammary human adipocytes contain one of the highest monoamine oxidase activities in the body. Characterization of the enzyme isoforms by inhibition profiles of [14C]tyramine oxidation and Western and Northern blot analyses showed that mRNAs and proteins related to both monoamine oxidases A and B were expressed in adipocytes. Quantification of each enzyme isoform performed by enzyme assay and Western blot showed that monoamine oxidase A was predominant, representing 70-80% of the total enzyme activity. In uptake experiments, the monoamine oxidase substrate [3H]noradrenaline was transported into white adipocytes (Vmax 0.81+/-0.3 nmol/30 min/100 mg of lipid, Km 235+/-104 microM). The inhibition of [3H]noradrenaline uptake by specific inhibitors indicated that white human adipocytes contain an extraneuronal-type noradrenaline transporter. Competition studies of [14C]tyramine oxidation showed that noradrenaline is metabolized by monoamine oxidases in intact cells. In conclusion, the concomitant expression of monoamine oxidases and of a noradrenaline transporter in human white adipocytes supports the role of the adipose tissue in the clearance of peripheral catecholamines. These results suggest that adipocytes should be considered as a previously unknown potential target of drugs acting on monoamine oxidases and noradrenaline transporters.

Oral Insulin-Mimetic Compounds That Act Independently of Insulin

The hallmarks of insulin action are the stimulation and suppression of anabolic and catabolic responses, respectively. These responses are orchestrated by the insulin pathway and are initiated by the binding of insulin to the insulin receptor, which leads to activation of the receptor’s intrinsic tyrosine kinase. Severe defects in the insulin pathway, such as in types A and B and advanced type 1 and 2 diabetes lead to severe insulin resistance, resulting in a partial or complete absence of response to exogenous insulin and other known classes of antidiabetes therapies. We have characterized a novel class of arylalkylamine vanadium salts that exert potent insulin-mimetic effects downstream of the insulin receptor in adipocytes. These compounds trigger insulin signaling, which is characterized by rapid activation of insulin receptor substrate-1, Akt, and glycogen synthase kinase-3 independent of insulin receptor phosphorylation. Administration of these compounds to animal models of diabetes lowered glycemia and normalized the plasma lipid profile. Arylalkylamine vanadium compounds also showed antidiabetic effects in severely diabetic rats with undetectable circulating insulin. These results demonstrate the feasibility of insulin-like regulation in the complete absence of insulin and downstream of the insulin receptor. This represents a novel therapeutic approach for diabetic patients with severe insulin resistance.

α2-adrenoceptors in the HT 29 human colon adenocarcinoma cell line: characterization with [3H]clonidine; effects on cyclic AMP accumulation

In the present work, [3H]clonidine was used to characterize alpha 2-adrenoceptors on the human adenocarcinoma cell line HT 29. The effects of alpha 2-adrenergic stimulation on cellular cyclic AMP levels were also investigated. The binding of [3H]clonidine on HT 29 cell membrane preparations was rapid and reversible. Scatchard analysis of the saturation curves indicated the existence of a single class of non-interacting sites with a KD of 1.29 +/- 0.07 nM and a Bmax of 114 +/- 7 fmol/mg of cell membrane protein. The binding sites for [3H]clonidine showed the required specificity for alpha 2-adrenoceptors. The potencies of alpha-adrenergic compounds to displace [3H]clonidine binding ranked as follows: yohimbine greater than phentolamine much greater than prazosin for antagonists and clonidine greater than epinephrine greater than norepinephrine greater than phenylephrine much greater than amidephrine for agonists. When tested on intact cells, epinephrine, norepinephrine and clonidine were found to counteract, in a dose-dependent manner, the increase of cyclic AMP triggered by vasoactive intestinal peptide (VIP). Such inhibitory effects were abolished by the addition of yohimbine but not of prazosin. The physiological amines were the most efficient agonists: both epinephrine and norepinephrine inhibited VIP-induced cyclic AMP accumulation by 50-55% with KD values of 50 nM and 300 nM respectively. Clonidine was a partial agonist only, provoking a weak (25-30%) inhibition of VIP-induced cyclic AMP accumulation even at high concentrations. These results indicate that, like normal colocytes, human colon adenocarcinoma cells HT 29 possess alpha 2-adrenoceptors, the stimulation of which is associated with an inhibition of cyclic AMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Imidazolinic radioligands for the identification of hamster adipocyte α2-adrenoceptors

Imidazolinic radioligands ([3H]UK 14304, [3H]idazoxan and [3H]RX 821002) were used for the identification of alpha 2-adrenoceptors on hamster fat cell membranes since there are limitations to the use of [3H]yohimbine and [3H]clonidine, which suggest alpha 2-adrenoceptor heterogeneity. Biological assays (lipolysis measurements) were performed on isolated fat cells and binding studies were carried out on fat cell membranes. The imidazolinic derivative, UK 14304, was a full agonist as compared to clonidine. Idazoxan and RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline), a recently developed alpha 2-antagonist, were more potent alpha 2-antagonists than yohimbine in this fat cell model. [3H]UK 14304 was the most suitable agent for the quantification of the high-affinity state alpha 2-adrenoceptors in binding studies since it did not exhibit the sensitivity to the composition of the buffer shown by [3H]clonidine. Although it is a potent alpha 2-antagonist, [3H]idazoxan had major limitations for use in the identification of alpha 2-adrenoceptors in this cell model since it also bound to non-adrenaline displaceable binding sites which were revealed when imidazolinic derivatives (phentolamine) were used instead of adrenaline to determine the non-specific binding. We demonstrated that [3H]RX 821002 was a more suitable radioligand than [3H]yohimbine for labelling hamster fat cell alpha 2-adrenoceptors (KD = 1.0 +/- 0.1 nM, Bmax = 776 +/- 60 fmol/mg protein). Moreover, since it exhibited low affinity for imidazoline-preferring sites, it represents a valuable ligand even in tissues possessing such binding sites. We suggest that [3H]RX 821002 can be used to identify alpha 2-adrenoceptors in various tissues when these sites cannot be labelled with [3H]yohimbine.

Lack of functional antilipolytic α-adrenoceptor in rat fat cell: Comparison with hamster adipocyte

Abstract 1. The small population of [ 3 H]clonidine binding sites in rat fat cell membranes do not have the characteristics of typical α 2 -adrenoceptors. 2. Clonidine (an α 2 -adrenoceptor agonist) has no antilipolytic effect on rat fat cells stimulated by theophylline. 3. In contrast to the rat, [ 3 H]clonidine labels an α 2 -adrenoceptor in hamster fat cell membranes and clonidine exerts a strong antilipolytic effect on theophylline-stimulated lipolysis.