Quantitative 3D comparison of biofilm imaged by X-ray micro-tomography and two-photon laser scanning microscopy.

Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X-ray micro-tomography (X-ray CMT) might be an alternative but it finds limitations in similarity of X-ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high-resolution 3D imaging of biofilm via X-ray CMT. However, this approach lacks comparison with well-established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two-photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X-ray CMT using an improved protocol where barium sulphate is combined with low-gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono-dispersed fluorescent and X-ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X-ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM.

Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin.

BACKGROUND/OBJECTIVES: Impaired energy metabolism is the defining characteristic of obesity-related heart failure. The adipocyte-derived peptide apelin has a role in the regulation of cardiovascular and metabolic homeostasis and may contribute to the link between obesity, energy metabolism and cardiac function. Here we investigate the role of apelin in the transition from metabolic adaptation to maladaptation of the heart in obese state. METHODS: Adult male C57BL/6J, apelin knock-out (KO) or wild-type mice were fed a high-fat diet (HFD) for 18 weeks. To induce heart failure, mice were subjected to pressure overload after 18 weeks of HFD. Long-term effects of apelin on fatty acid (FA) oxidation, glucose metabolism, cardiac function and mitochondrial changes were evaluated in HFD-fed mice after 4 weeks of pressure overload. Cardiomyocytes from HFD-fed mice were isolated for analysis of metabolic responses. RESULTS: In HFD-fed mice, pressure overload-induced transition from hypertrophy to heart failure is associated with reduced FA utilization (P<0.05), accelerated glucose oxidation (P<0.05) and mitochondrial damage. Treatment of HFD-fed mice with apelin for 4 weeks prevented pressure overload-induced decline in FA metabolism (P<0.05) and mitochondrial defects. Furthermore, apelin treatment lowered fasting plasma glucose (P<0.01), improved glucose tolerance (P<0.05) and preserved cardiac function (P<0.05) in HFD-fed mice subjected to pressure overload. In apelin KO HFD-fed mice, spontaneous cardiac dysfunction is associated with reduced FA oxidation (P<0.001) and increased glucose oxidation (P<0.05). In isolated cardiomyocytes, apelin stimulated FA oxidation in a dose-dependent manner and this effect was prevented by small interfering RNA sirtuin 3 knockdown. CONCLUSIONS: These data suggest that obesity-related decline in cardiac function is associated with defective myocardial energy metabolism and mitochondrial abnormalities. Furthermore, our work points for therapeutic potential of apelin to prevent myocardial metabolic abnormalities in heart failure paired with obesity.

Impairment of adipose tissue in Prader-Willi syndrome rescued by growth hormone treatment.

BACKGROUND: Prader-Willi syndrome (PWS) results from abnormalities in the genomic imprinting process leading to hypothalamic dysfunction with an alteration of growth hormone (GH) secretion. PWS is associated with early morbid obesity and short stature which can be efficiently improved with GH treatment. OBJECTIVES: Our aims were to highlight adipose tissue structural and functional impairments in children with PWS and to study the modifications of those parameters on GH treatment. SUBJECTS AND METHODS: Plasma samples and adipose tissue biopsies were obtained from 23 research centers in France coordinated by the reference center for PWS in Toulouse, France. Lean controls (n=33), non-syndromic obese (n=53), untreated (n=26) and GH-treated PWS (n=43) children were enrolled in the study. Adipose tissue biopsies were obtained during scheduled surgeries from 15 lean control, 7 untreated and 8 GH-treated PWS children. RESULTS: Children with PWS displayed higher insulin sensitivity as shown by reduced glycemia, insulinemia and HOMA-IR compared with non-syndromic obese children. In contrast, plasma inflammatory cytokines such as TNF-α, MCP-1 and IL-8 were increased in PWS. Analysis of biopsies compared with control children revealed decreased progenitor cell content in the stromal vascular fraction of adipose tissue and an impairment of lipolytic response to ß-adrenergic agonist in PWS adipocytes. Interestingly, both of these alterations in PWS seem to be ameliorated on GH treatment. CONCLUSION: Herein, we report adipose tissue dysfunctions in children with PWS which may be partially restored by GH treatment.

Effect of endurance training on skeletal muscle myokine expression in obese men: identification of apelin as a novel myokine.

BACKGROUND: It has been suggested that the metabolic benefits of physical exercise could be mediated by myokines. We examined here the effect of exercise training on skeletal muscle expression of a panel of myokines in humans. Pathways regulating myokine expression were investigated in human myotubes. METHODS: Eleven obese non-diabetic male subjects were enrolled in an 8-week endurance training program. Insulin sensitivity was assessed by an oral glucose tolerance test. Subcutaneous adipose tissue and Vastus lateralis muscle biopsy samples were collected before and after training. RNAs were prepared from adipose tissue and skeletal muscle. Primary culture of myoblasts was established. RESULTS: As expected, exercise training improved aerobic capacity and decreased fat mass. No significant change in interleukin 6, fibroblast growth factor 21, myostatin (MSTN) or irisin mRNA level was found in muscle after training. A twofold increase in apelin mRNA level was found in muscle but not in adipose tissue. No change in circulating myokine and adipokine plasma levels was observed in the resting state in response to training. Interestingly, apelin was significantly expressed and secreted in primary human myotubes. Apelin gene expression was upregulated by cyclic AMP and calcium, unlike the other myokines investigated. Importantly, changes in muscle apelin mRNA levels were positively related to whole-body insulin sensitivity improvement. CONCLUSION: Collectively, our data show that exercise training upregulates muscle apelin expression in obese subjects. Apelin expression is induced by exercise signaling pathways and secreted in vitro in human primary myotubes, and may behave as a novel exercise-regulated myokine with autocrine/paracrine action.

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 (20  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.

Altered food consumption in mice lacking lysophosphatidic acid receptor-1

No disponible

The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production (AU)

Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

Caffeine reduces TNFá up-regulation in humanadipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesityrelatedpathologies such as diabetes. Through inflammatory cytokines production,adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promoteadipocyte insulin resistance by a paracrine way. Since xanthine family compoundssuch as caffeine were shown to decrease inflammatory production by humanblood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factorá (TNFá) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture.For that purpose, human subcutaneous adipose tissue obtained from healthynon-obese women (BMI: 26.7 ± 2.2 kg/m2) after abdominal dermolipectomy, wassplit into explants and cultured for 6 hours with or without caffeine. Three differentconcentrations of caffeine were tested (0.5ìg/mL, 5ìg/mL and 50ìg/mL). After 6hours of treatment, explants were subjected to collagenase digestion in order to isolateadipocytes and SVF cells. Then, TNFá and IL-6 mRNA were analysed by realtimePCR alternatively in adipocytes and SVF cells. In parallel, we checked geneexpression of markers involved in adipocyte differenciation and in SVF cells inflammationand proliferation. Our findings show a strong and dose dependent down-regulationof TNF-á gene expression in both adipocyte and SVF cells whereas IL-6 wasonly down regulated in SVF cells. No effect of caffeine was noticed on the othergenes studied. Thus, caffeine, by decreasing TNFá expression, could improve adiposetissue inflammation during obesity (AU)

No disponible

Caffeine reduces TNFalpha up-regulation in human adipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesityrelatedpathologies such as diabetes. Through inflammatory cytokines production,adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promoteadipocyte insulin resistance by a paracrine way. Since xanthine family compoundssuch as caffeine were shown to decrease inflammatory production by humanblood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factoralpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture.For that purpose, human subcutaneous adipose tissue obtained from healthynon-obese women (BMI: 26.7 ± 2.2 kg/m2) after abdominal dermolipectomy, wassplit into explants and cultured for 6 hours with or without caffeine. Three differentconcentrations of caffeine were tested (0.5ìg/mL, 5ìg/mL and 50ìg/mL). After 6hours of treatment, explants were subjected to collagenase digestion in order to isolateadipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by realtimePCR alternatively in adipocytes and SVF cells. In parallel, we checked geneexpression of markers involved in adipocyte differenciation and in SVF cells inflammationand proliferation. Our findings show a strong and dose dependent down-regulationof TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 wasonly down regulated in SVF cells. No effect of caffeine was noticed on the othergenes studied. Thus, caffeine, by decreasing TNFá expression, could improve adiposetissue inflammation during obesity (AU)

No disponible

Alterations of lipid metabolism and gene expression in rat adipocytes during chronic olanzapine treatment.

Although antipsychotics are established drugs in schizophrenia treatment, they are admittedly known to induce side effects favoring the onset of obesity and worsening its complications. Despite potential involvement of histamine receptor antagonism, or of other neurotransmitter systems, the mechanism by which antipsychotic drugs increase body weight is not elucidated. The aim of the present study was to investigate whether chronic antipsychotic treatments can directly alter the regulation of two main functions of white adipose tissue: lipolysis and glucose utilization. The influence of a classical antipsychotic (haloperidol) was compared to that of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (ziprasidone). Cell size, lipolytic capacity and glucose transport activity were determined in white adipocytes of rats subjected to 5-week oral treatment with these antipsychotics. Gene expression of adipocyte proteins involved in glucose transport or fat storage and mobilization, such as glucose transporters (GLUT1 and GLUT4), leptin, matrix metallo-proteinase-9 (MMP9), hormone-sensitive lipase (HSL) and fatty acid synthase (FAS) was also evaluated. Adipocytes from chronic olanzapine-treated rats exhibited decreased lipolytic activity, lowered HSL expression and increased FAS expression. These changes were concomitant to enlarged fat deposition and adipocyte size. Alterations were observed in adipocytes from olanzapine-treated rats whereas the other antipsychotics did not induce any notable disorder. Our results therefore show evidence of an effect of chronic antipsychotic treatment on rat adipocyte metabolism. Thus, impairment of fat cell lipolysis should be considered as a side effect of certain antipsychotics, leading, along with the already documented hyperphagia, to the excessive weight gain observed in patients under prolonged treatment..

Caffeine reduces TNFalpha up-regulation in human adipose tissue primary culture.

Adipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity.

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.

Effects of oral administration of benzylamine on glucose tolerance and lipid metabolism in rats.

Repeated administration of benzylamine plus vanadate have been reported to exhibit anti-hyperglycemic effects in different models of diabetic rats. Likewise oral treatment with Moringa oleifera extracts which contain the alkaloïd moringine, identical to benzylamine, has also been shown to prevent hyperglycemia in alloxan-induced diabetic rats. With these observations we tested whether prolonged oral administration of benzylamine could interact with glucose and/or lipid metabolism. Seven week old male Wistar rats were treated for seven weeks with benzylamine 2.9 g/l in drinking water and were submitted to glucose tolerance tests. A slight decrease in water consumption was observed in benzylamine-treated animals while there was no change in body and adipose tissue weights at the end of treatment. Blood glucose and plasma insulin, triacylglycerol or cholesterol levels were not modified. However, benzylamine treatment resulted in a decrease in plasma free fatty acids in both fed and fasted conditions. Benzylamine treatment improved glucose tolerance as shown by the reduction of hyperglycemic response to intra-peritoneal glucose load. Oral benzylamine treatment did not alter the response of adipocytes to insulin nor to insulin-like actions of benzylamine plus vanadate, via in vitro activation of glucose transport or inhibition of lipolysis. This work demonstrates for the first time that oral administration of benzylamine alone influences glucose and lipid metabolism. However, these results obtained in normoglycemic rats require to be confirmed in diabetic models.

Effects of oral administration of benzylamine on glucose tolerance and lipid metabolism in rats

Repeated administration of benzylamine plus vanadate have been reported to exhibit anti-hyperglycemic effects in different models of diabetic rats. Likewise oral treatment with Moringa oleifera extracts which contain the alkaloïd moringine, identical to benzylamine, has also been shown to prevent hyperglycemia in alloxan-induced diabetic rats. With these observations we tested whether prolonged oral administration of benzylamine could interact with glucose and/or lipid metabolism. Seven week old male Wistar rats were treated for seven weeks with benzylamine 2.9 g/l in drinking water and were submitted to glucose tolerance tests. A slight decrease in water consumption was observed in benzylamine-treated animals while there was no change in body and adipose tissue weights at the end of treatment. Blood glucose and plasma insulin, triacylglycerol or cholesterol levels were not modified. However, benzylamine treatment resulted in a decrease in plasma free fatty acids in both fed and fasted conditions. Benzylamine treatment improved glucose tolerance as shown by the reduction of hyperglycemic response to intra-peritoneal glucose load. Oral benzylamine treatment did not alter the response of adipocytes to insulin nor to insulin-like actions of benzylamine plus vanadate, via in vitro activation of glucose transport or inhibition of lipolysis. This work demonstrates for the first time that oral administration of benzylamine alone influences glucose and lipid metabolism. However, these results obtained in normoglycemic rats require to be confirmed in diabetic models

En ratas diabéticas, la administración crónica de la combinación benzilamina más vanadato ejerce un efecto antidiabético. Recientemente se ha descrito en ratas diabéticas inducidas por aloxan una reducción de la glucemia tras el tratamiento oral con extracto de Moringa oleifera, que contiene el alcaloide moringina, idéntico a la benzilamina. Por ello, se investiga en este trabajo el efecto del tratamiento prolongado por via oral con sólo benzilamina sobre el metabolismo de la glucosa y/o los lípidos. Ratas macho Wistar de 7 semanas se trataron durante 7 semanas con benzilamina 2.9 g/l en el agua de la bebida. Al finalizar el tratamiento, las ratas fueron sometidos a un test de tolerancia a la glucosa, inyectada por via intraperitoneal. Se recogió plasma para la determinación bioquímica y se aislaron adipocitos para estudiar la lipólisis y la captación de glucosa. El tratamiento oral con benzilamina no modifica el peso corporal ni el de la grasa, ni los niveles plasmáticos de glucosa, insulina, triacilglicerol y colesterol. Sin embargo, mejora la tolerancia a la glucosa, pues reduce la respuesta hiperglucémica a la inyección intra-peritoneal de glucosa y reduce los niveles de acidos grasos, tanto en situación de ayuno como tras la ingesta. El tratamiento oral con benzilamina no modifica en el adipocito los efectos de insulina o benzilamina más vanadato sobre la activación del transporte de glucosa o la inhibición de la lipolisis. Este trabajo demuestra por vez primera que la administración oral de benzilamina influye sobre el metabolismo de los lípidos y de la glucosa. Sin embargo, estos resultados obtenidos en ratas normoglicémicas deben ser confirmados en modelos diabéticos

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.

Effect of non-digestible gluco-oligosaccharides on glucose sensitivity in high fat diet fed mice.

Non digestible dietary carbohydrates have been reported to modify lipaemia and post-prandial glycaemia and insulinaemia. The aim of this study was to investigate the effect of a non-digestible gluco-oligosaccharides (GOS) diet on glucose, insulin, triglycerides and free fatty acid blood levels and glucose sensitivity in high fat diet fed mice (a high fat diet composed of 45% fat, 35% carbohydrate and 20% protein). Female C57B16/J mice were divided into two groups fed a high fat diet (HF) for 20 weeks supplemented or not with 1.5 g/kg/day of GOS (HF-GOS). The GOS supplementation did not change body weight nor fat pad mass, nor any of the blood parameters measured (glucose, insulin, leptin, triglycerides, and free fatty acids). However, mice which received the GOS supplemented diet showed an increased glucose utilization after a 1 g/kg load of glucose compared with the mice fed the high fat diet alone. Our results suggest a role for non-digestible GOS in the regulation of carbohydrate metabolism.

[Development of nutritional obesity in transgenic mice with an adrenergic receptivity in adipose tissue comparable with that of humans]. / Développement d'une obésité nutritionnelle chez des souris transgéniques ayant une réceptivité adrénergique du tissu adipeux comparable à celle de l'homme.

Obesity is characterized by an excessive development of fat mass which is a consequence of increased fat cell size and/or fat cell number. Several hormones and neurotransmitters are regulators of adipose tissue development and metabolism. Among them, catecholamines play a major role by acting through alpha 2- and beta-adrenergic receptors. Stimulation of alpha 2-adrenergic receptors induce inhibition of lipolysis in mature adipocytes as well as preadipocyte proliferation. The antilipolytic effect mediated by alpha 2-adrenergic receptors is in part responsible for the weak lipid mobilization of some fat deposits in humans (subcutaneous fat in particular). Changes in beta- and alpha 2-adrenergic receptors ratio and function have been proposed to explain the lipolytic disturbances described in some obese subjects. Human and rodent adipocytes differ considerably with respect to the balance between beta- and alpha 2-adrenergic receptors. Human adipocytes express mainly alpha 2- but very few beta 3-adrenergic receptors while the reverse is true for rodent adipocytes. Since no suitable animal model was available to study the contribution of alpha 2/beta-adrenergic balance in adipocytes in vivo, we combined gene targeting and transgenic approaches to create a mice with increased alpha 2/beta-adrenergic ratio in adipose tissue. Specifically, we have generated transgenic mice strains on a beta 3-adrenergic receptor knock-out background which express human alpha 2-adrenergic receptors. No particular phenotype was observed in mice maintained in normal diet whereas when fed a high fat diet, transgenic mice increased significantly body weight and fat mass. These results underline the physiologic relevance of the interaction of the presence of alpha 2-adrenergic receptors with a high fat diet in the control of adipose tissue development.

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.

Endothelial differentiation gene-2 receptor is involved in lysophosphatidic acid-dependent control of 3T3F442A preadipocyte proliferation and spreading.

EDG-2, EDG-4, EDG-7, and PSP24 genes encode distinct lysophosphatidic acid (LPA) receptors. The aim of the present study was to determine which receptor subtype is involved in the biological responses generated by LPA in preadipocytes. Growing 3T3F442A preadipocytes express EDG-2 and EDG-4 mRNAs, with no expression of EDG-7 or PSP24 mRNAs. Quantitative reverse transcriptase-polymerase chain reaction revealed that EDG-2 transcripts were 10-fold more abundant than that of EDG-4. To determine the involvement of the EDG-2 receptor in the responses of growing preadipocytes to LPA, stable transfection of antisense EDG-2 cDNA was performed in growing 3T3F442A preadipocytes. This procedure, led to a significant and specific reduction in EDG-2 mRNA and protein. This was associated with a significant alteration in the effect of LPA on both cell proliferation and cell spreading. Finally, the differentiation of growing preadipocytes into quiescent adipocytes led to a strong reduction in the level of EDG-2 transcripts. Results demonstrate the significant contribution of the EDG-2 receptor in the biological responses generated by LPA in 3T3F442A preadipocytes.

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.