Combination of low dose of the anti-adipogenic agents resveratrol and phenelzine in drinking water is not sufficient to prevent obesity in very-high-fat diet-fed mice.

PURPOSE: Resveratrol inhibits lipid accumulation but suffers from limited bioavailability. The anti-depressive agent phenelzine limits adipogenesis in various models of cultured preadipocytes, and this hydrazine derivative also inhibits de novo lipogenesis in mature adipocytes. It was therefore tested whether resveratrol effects on adiposity reduction and glucose tolerance improvement could be reinforced by co-administration with phenelzine. METHODS: Mice fed a very-high-fat diet (VHFD, 60% calories as fat) were subjected to drinking solution containing low dose of resveratrol (0.003%) and/or 0.02% phenelzine for 12 weeks. Body fat content, glucose tolerance, food and water consumption were checked during treatment while fat depot mass was determined at the end of supplementation. Direct influence of the agents on lipogenesis and glucose uptake was tested in adipocytes. RESULTS: Epididymal fat depots were reduced in mice drinking phenelzine alone or with resveratrol. No limitation of body weight gain or body fat content was observed in the groups drinking resveratrol or phenelzine, separately or in combination. The altered glucose tolerance and the increased fat body composition of VHFD-fed mice were not reversed by resveratrol and/or phenelzine. Such lack of potentiation between resveratrol and phenelzine prompted us to verify in vitro their direct effects on mouse adipocytes. Both molecules inhibited de novo lipogenesis, but did not potentiate each other at 10 or 100 µM. Only resveratrol inhibited hexose uptake in a manner that was not improved by phenelzine. CONCLUSIONS: Phenelzine has no interest to be combined with low doses of resveratrol for treating/preventing obesity, when considering the VHFD mouse model.

Lysophosphatidic acid impairs glucose homeostasis and inhibits insulin secretion in high-fat diet obese mice.

AIMS/HYPOTHESIS: Lysophosphatidic acid (LPA) is a lipid mediator produced by adipocytes that acts via specific G-protein-coupled receptors; its synthesis is modulated in obesity. We previously reported that reducing adipocyte LPA production in high-fat diet (HFD)-fed obese mice is associated with improved glucose tolerance, suggesting a negative impact of LPA on glucose homeostasis. Here, our aim was to test this hypothesis. METHODS: First, glucose tolerance and plasma insulin were assessed after acute (30 min) injection of LPA (50 mg/kg) or of the LPA1/LPA3 receptor antagonist Ki16425 (5 mg kg(-1) day(-1), i.p.) in non-obese mice fed a normal diet (ND) and in obese/prediabetic (defined as glucose-intolerant) HFD mice. Glucose and insulin tolerance, pancreas morphology, glycogen storage, glucose oxidation and glucose transport were then studied after chronic treatment (3 weeks) of HFD mice with Ki16425. RESULTS: In ND and HFD mice, LPA acutely impaired glucose tolerance by inhibiting glucose-induced insulin secretion. These effects were blocked by pre-injection of Ki16425 (5 mg/kg, i.p.). Inhibition of glucose-induced insulin secretion by LPA also occurred in isolated mouse islets. Plasma LPA was higher in HFD mice than in ND mice and Ki16425 transiently improved glucose tolerance. The beneficial effect of Ki16425 became permanent after chronic treatment and was associated with increased pancreatic islet mass and higher fasting insulinaemia. Chronic treatment with Ki16425 also improved insulin tolerance and increased liver glycogen storage and basal glucose use in skeletal muscle. CONCLUSIONS/INTERPRETATION: Exogenous and endogenous LPA exerts a deleterious effect on glucose disposal through a reduction of plasma insulin; pharmacological blockade of LPA receptors improves glucose homeostasis in obese/prediabetic mice.

Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL.

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Histamine oxidation in mouse adipose tissue is controlled by the AOC3 gene-encoded amine oxidase.

INTRODUCTION: Histaminergic status can modify adipose tissue (AT) development: histamine-free mice exhibit visceral obesity, and treatments with H3-antagonists reduce body weight gain. However, direct histamine effects on AT remain poorly documented: it has been observed that histamine stimulates lipolysis in rodent adipocytes when its oxidation by amine oxidases (AOs) is blocked by inhibitors such as semicarbazide. OBJECTIVE: The aim of this work was to study the influence of AOC3 gene invalidation, encoding for semicarbazide-sensitive AO (SSAO), on histamine oxidation and on histamine lipolytic activity in AT. MATERIALS AND METHODS: Expression of AOC- and MAO-encoding genes was determined by real-type PCR in wild-type (WT) and SSAO-deficient (AOC3-KO) mice. Lipolysis was assessed by glycerol release in isolated adipocytes and AO activity by substrate-induced hydrogen peroxide formation in kidney, ileum and AT. RESULTS: The expression levels of the genes encoding AOC1, AOC2 or MAOA and MAOB were not modified in the AT of AOC3-KO mice. In WT mice, histamine oxidation was lower than that of the reference SSAO-substrate benzylamine in AT, but not in ileum. The order of magnitude regarding benzylamine oxidation was AT > ileum >> kidney. In AOC3-KO mice, benzylamine oxidation was abolished in all tissues, while histamine oxidation was abolished in AT but not in ileum. Histamine was inactive on lipolysis in WT but stimulated lipolysis in fat cells from AOC3-KO mice, without reaching the maximal intensity of beta-adrenergic stimulation. CONCLUSION: Histamine was mainly oxidized by diamine oxidase (AOC1 product) in intestine, but by SSAO (AOC3 product) in AT. When protected from its oxidation by SSAO in AT, histamine moderately activated lipolysis in adipocytes in AOC3-KO mice.

Altered food consumption in mice lacking lysophosphatidic acid receptor-1

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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.

Influence of acute and chronic administration of benzylamine on glucose tolerance in diabetic and obese mice fed on very high-fat diet

The combination of vanadate plus benzylamine has been reported to stimulateglucose transport in rodent adipocytes and to mimic other insulin actions in diversestudies. However, benzylamine alone activates glucose uptake in human fat cells andincreases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamineantihyperglycemic action and to test whether its chronic oral administrationcould restore the defective glucose handling of mice rendered slightly obese anddiabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected withbenzylamine at 0.7 to 700 ìmol/kg before glucose tolerance test, they exhibitedreduced hyperglycemic response without alteration of insulin secretion. Whole bodyglucose turnover, as assessed by the glucose isotopic dilution technique, wasunchanged in mice perfused with benzylamine (total dose of 75 ìmol/kg). However,their in vivo glycogen synthesis rate was increased. Benzylamine appeared thereforeto directly facilitate glucose utilisation in peripheral tissues. When given chronicallyat 2000 or 4000 ìmol/kg/d in drinking water, benzylamine elicited a slightreduction of water consumption but did not change body weight or adiposity anddid not modify oxidative stress markers. Benzylamine treatment improved glucosa tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFDmice. There was no influence of benzylamine ingestion on lipolytic activity, basal andinsulin-stimulated glucose uptake, and on inflammatory adipokine expression inadipocytes. The improvement of glucose tolerance and the lack of adverse effects onadipocyte metabolism, reported here in VHFD mice allow to consider orally givenbenzylamine as a potential antidiabetic strategy which deserves to be further studied in other diabetic models (AU)

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Influence of acute and chronic administration ofbenzylamine on glucose tolerance in diabetic andobese mice fed on very high-fat diet

The combination of vanadate plus benzylamine has been reported to stimulateglucose transport in rodent adipocytes and to mimic other insulin actions in diversestudies. However, benzylamine alone activates glucose uptake in human fat cells andincreases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamineantihyperglycemic action and to test whether its chronic oral administrationcould restore the defective glucose handling of mice rendered slightly obese anddiabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected withbenzylamine at 0.7 to 700 ìmol/kg before glucose tolerance test, they exhibitedreduced hyperglycemic response without alteration of insulin secretion. Whole bodyglucose turnover, as assessed by the glucose isotopic dilution technique, wasunchanged in mice perfused with benzylamine (total dose of 75 ìmol/kg). However,their in vivo glycogen synthesis rate was increased. Benzylamine appeared thereforeto directly facilitate glucose utilisation in peripheral tissues. When given chronicallyat 2000 or 4000 ìmol/kg/d in drinking water, benzylamine elicited a slightreduction of water consumption but did not change body weight or adiposity anddid not modify oxidative stress markers. Benzylamine treatment improved glucose tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFDmice. There was no influence of benzylamine ingestion on lipolytic activity, basal andinsulin-stimulated glucose uptake, and on inflammatory adipokine expression inadipocytes. The improvement of glucose tolerance and the lack of adverse effects onadipocyte metabolism, reported here in VHFD mice allow to consider orally givenbenzylamine as a potential antidiabetic strategy which deserves to be further studiedin other diabetic models (AU)

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Influence of acute and chronic administration of benzylamine on glucose tolerance in diabetic and obese mice fed on very high-fat diet.

The combination of vanadate plus benzylamine has been reported to stimulate glucose transport in rodent adipocytes and to mimic other insulin actions in diverse studies. However, benzylamine alone activates glucose uptake in human fat cells and increases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamine antihyperglycemic action and to test whether its chronic oral administration could restore the defective glucose handling of mice rendered slightly obese and diabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected with benzylamine at 0.7 to 700 micromol/kg before glucose tolerance test, they exhibited reduced hyperglycemic response without alteration of insulin secretion. Whole body glucose turnover, as assessed by the glucose isotopic dilution technique, was unchanged in mice perfused with benzylamine (total dose of 75 micromol/kg). However, their in vivo glycogen synthesis rate was increased. Benzylamine appeared therefore to directly facilitate glucose utilisation in peripheral tissues. When given chronically at 2000 or 4000 micromol/kg/d in drinking water, benzylamine elicited a slight reduction of water consumption but did not change body weight or adiposity and did not modify oxidative stress markers. Benzylamine treatment improved glucose tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFD mice. There was no influence of benzylamine ingestion on lipolytic activity, basal and insulin-stimulated glucose uptake, and on inflammatory adipokine expression in adipocytes. The improvement of glucose tolerance and the lack of adverse effects on adipocyte metabolism, reported here in VHFD mice allow to consider orally given benzylamine as a potential antidiabetic strategy which deserves to be further studied in other diabetic models.

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.

The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.

The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment.

Thrombin, the terminal serine protease in the coagulation cascade, is a proinflammatory molecule in vivo and induces endothelial activation in vitro. The cellular signaling mechanisms involved in this function are unknown. The role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in thrombin-induced chemokine production was studied. Phosphorylation of both p38 MAPK and its substrate, ATF-2, was observed in human umbilical vein endothelial cells (HUVECs) stimulated with thrombin, with a maximum after 5 minutes of stimulation. Using the selective p38 MAPK inhibitor SB203580, there was a significant decrease in thrombin-induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) protein production and messenger RNA steady-state levels. In addition, SB203580 decreased IL-8 and MCP-1 production induced by the thrombin receptor-1 agonist peptide (TRAP), suggesting functional links between the thrombin G protein-coupled receptor and the p38 MAPK pathway. Furthermore, endothelial activation in the presence of SB203580 decreased the chemotactic activity of thrombin-stimulated HUVEC supernatant on neutrophils and monocytic cells. In contrast, the p42/p44 MAPK pathway did not appear to be involved in thrombin- or TRAP-induced endothelial chemokine production, because there was no reduction in the presence of the p42/p44-specific inhibitor PD98059. These results demonstrate that the p38 rather than p42/44 MAPK signaling pathway plays an important role in thrombin-induced endothelial proinflammatory activation and suggest that inhibition of p38 MAPK may be an interesting target for anti-inflammatory strategies in vascular diseases combining thrombosis and inflammation. (Blood. 2001;98:667-673)

Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells.

Endothelial cells play a key role in prominent immunological and pathological processes such as leukocyte trafficking, inflammation, atheroma or cancer cell metastasis. Umbilical veins are probably the most widely used source for human endothelial cells, since they are more easily available than many other vessels, they are free from any pathological process and they are physiologically more relevant than many established cell lines. Here, we describe a standard protocol for preparation, maintenance and quality control of these cells.

Dietary soybean phosphatidylcholines lower lipidemia: mechanisms at the levels of intestine, endothelial cell, and hepato-biliary axis.

The beneficial metabolic effects of dietary soybean lecithin on lipid metabolism are now more clearly established. The intestinal absorption of cholesterol is decreased by soybean phosphatidylcholine-enriched diet and results in a cholesterol-lowering effect. There is an enhancement of the cholesterol efflux by endothelial cells incubated with soybean phosphatidylcholines, and a stimulation of the reverse cholesterol transport by high density lipoprotein-phosphatidylcholines. As a result of all these processes, phosphatidylcholines provided by the soybean lecithin metabolism appear to be key molecules controlling the biodynamic exchanges of lipids. They regulate homeostasis of cholesterol and fatty acids by decreasing their synthesis and promoting cholesterol oxidation into bile salts. Finally, the outcome is the increase in bile secretion of these lipids and/or their metabolite forms. Such findings constitute promising goals in the field of nutritional effects of soybean lecithin in the treatment or prevention of hyperlipidemia and related atherosclerosis.