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Whereas reactive oxygen species (ROS) can have opposite impacts on insulin signaling, they have mainly been associated with mitochondrial dysfunction in skeletal muscle. We analyzed the relationship between these three features in skeletal muscle of senescence accelerated mice (SAM) prone (P8), which are characterized by enhanced oxidative stress compared to SAM resistant (R1). Oxidative stress, ROS production, antioxidant system, mitochondrial content and functioning, as well as in vitro and in vivo insulin signaling were investigated in gastrocnemius and quadriceps muscles. In SAMP8 compared to SAMR1, muscle content in carbonylated proteins was two-fold (p < 0.01) and ROS production by xanthine oxidase 70% (p < 0.05) higher. Furthermore, insulin-induced Akt phosphorylation measured in vivo and ex vivo as well as muscle glucose uptake measured ex vivo were significantly higher (p < 0.05). Mitochondrial respiration evidenced uncoupling and higher respiration rates with substrates of complexes II and IV, in agreement with higher maximal activity of complexes II and IV (+ 18% and 62%, respectively, p < 0.05). By contrast, maximal activity of complex I was 22% lower (p < 0.05). All strain differences were corrected after 6 months of N-acetylcysteine (NAC) treatment, thus supporting the involvement of high ROS production in these differences. In conclusion in muscle of SAMP8 compared to SAMR1, high ROS production is associated to higher insulin sensitivity and glucose uptake but to lower mitochondrial complex I activity. These conflicting adaptations, with regards to the resulting imbalance between NADH production and use, were associated with intrinsic adjustments in the mitochondrial respiration chain (mitochondrial uncoupling, enhanced complexes II and IV activity). We propose that these bioenergetics adaptations may help at preserving muscle metabolic flexibility of SAMP8.

Glucotoxicity-induced β-cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and the endoplasmic reticulum (ER). Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. The role of MAMs in β-cells is still largely unknown, and their implication in glucotoxicity-associated β-cell dysfunction remains to be defined. Here, we report that acute glucose treatment stimulated ER-mitochondria interactions and calcium (Ca

International audience; Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently shown to be involved in the control of hepatic insulin action and glucose homeostasis, but with conflicting results. Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alterations in insulin-resistant states, the relevance of MAM integrity in muscle insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin signaling could help to clarify this controversy. Here, we show in skeletal muscle of different mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria interactions as an early event preceding mitochondrial dysfunction and insulin resistance. Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a reduction of structural and functional ER-mitochondria interactions. Importantly, experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-induced alterations of insulin signaling and action, whereas disruption of MAM integrity alters the action of the hormone. Lastly, we found an association between altered insulin signaling and ER-mitochondria interactions in human myotubes from obese subjects with or without T2D compared with healthy lean subjects. Collectively, our data reveal a new role of MAM integrity in insulin action of skeletal muscle and highlight MAM disruption as an essential subcellular alteration associated with muscle insulin resistance in mice and humans. Therefore, reduced ER-mitochondria coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of obesity and T2D.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice.We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca(2+) exchange, organelle homeostasis and insulin action.Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca(2+) transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells.Disruption of IP3R-mediated Ca(2+) signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

International audience; Type 2 diabetes is characterized by peripheral insulin resistance and pancreatic beta cell dysfunction. Elevated free fatty acids (FFAs) may impair beta cell function and mass (lipotoxicity). Altered calcium homeostasis may be involved in defective insulin release. The endoplasmic reticulum (ER) is the major intracellular calcium store. Lipotoxicity induces ER stress and in parallel an ER calcium depletion through unknown ER calcium leak channels. The main purposes of this study is first to identify one of these channels and secondly, to check the opportunity to restore beta cells function (i.e., insulin secretion) after pharmacological inhibition of ER calcium store depletion. We investigated the functionality of translocon, an ER calcium leak channel and its involvement on FFAs-induced alterations in MIN6B1 cells and in human pancreatic islets. We evidenced that translocon acts as a functional ER calcium leak channel in human beta cells using anisomycin and puromycin (antibiotics), respectively blocker and opener of this channel. Puromycin induced a significant ER calcium release, inhibited by anisomycin pretreatment. Palmitate treatment was used as FFA model to induce a mild lipotoxic effect: ER calcium content was reduced, ER stress but not apoptosis were induced and glucose induced insulin secretion was decreased in our beta cells. Interestingly, translocon inhibition by chronic anisomycin treatment prevented dysfunctions induced by palmitate, avoiding reticular calcium depletion, ER stress and restoring insulin secretion. Our results provide for the first time compelling evidence that translocon actively participates to the palmitate-induced ER calcium leak and insulin secretion decrease in beta cells. Its inhibition reduces these lipotoxic effects. Taken together, our data indicate that TLC may be a new potential target for the treatment of type 2 diabetes.

ISI Document Delivery No.: 004QQTimes Cited: 0Cited Reference Count: 46Cited References: Pillon Nicolas J., 2011, CHEMICAL RESEARCH IN TOXICOLOGY, V24, P752Awada, Manar Soulage, Christophe O. Meynier, Anne Debard, Cyrille Plaisancie, Pascale Benoit, Berengere Picard, Gregory Loizon, Emmanuelle Chauvin, Marie-Agnes Estienne, Monique Peretti, Noel Guichardant, Michel Lagarde, Michel Genot, Claude Michalski, Marie-CarolineFrench National Research Agency (ANR)[ANR-08-ALIA-002]; ANRThis work was supported by the French National Research Agency (ANR) Grant ANR-08-ALIA-002, AGECANINOX project.M. Awada acknowledges her PhD grant from ANR. The authors thank Lucie Ribourg and Michele Viau for their work on oil mixtures. Patricia Daira is acknowledged for her help with the 4-hydroxy-2-alkenal assay. The authors thank Jean-Michel Chardigny for useful discussions. Isabelle Jouanin (Plateforme AXIOM-MetaToul, Toulouse) is acknowledged for the synthesis of internal standards to measure 4-hydroxyl-2-alkenals in fat. Yvonne Masson is acknowledged for revising the English-language manuscript.Amer soc biochemistry molecular biology incBethesda[Awada, Manar; Plaisancie, Pascale; Estienne, Monique; Michalski, Marie-Caroline] INRA, CarMeN Lab U1235, F-69621 Villeurbanne, France. [Awada, Manar; Soulage, Christophe O.; Guichardant, Michel; Lagarde, Michel; Michalski, Marie-Caroline] Inst Natl Sci Appl, IMBL, F-69621 Villeurbanne, France. [Meynier, Anne; Genot, Claude] INRA, Biopolymeres Interact Assemblages UR1268, F-44300 Nantes, France. [Debard, Cyrille; Chauvin, Marie-Agnes] INSERM, U1060, CarMeN Lab, F-69921 Oullins, France. [Benoit, Berengere; Picard, Gregory; Loizon, Emmanuelle; Peretti, Noel; Michalski, Marie-Caroline] Univ Lyon 1, F-69622 Villeurbanne, France.marie-caroline.michalski@insa-lyon.fr; International audience; Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

International audience; Endoplasmic reticulum (ER) stress is proposed as a novel link between elevated fatty acids levels, obesity and insulin resistance in liver and adipose tissue. However, it is unknown whether ER stress also contributes to lipid-induced insulin resistance in skeletal muscle, the major tissue responsible of insulin-stimulated glucose disposal. Here, we investigated the possible role of ER stress in palmitate-induced alterations of insulin action, both in vivo, in gastrocnemius of high-palm diet fed mice, and in vitro, in palmitate-treated C(2)C(12) myotubes. We demonstrated that 8 weeks of high-palm diet increased the expression of ER stress markers in muscle of mice, whereas ex-vivo insulin-stimulated PKB phosphorylation was not altered in this tissue. In addition, exposure of C(2)C(12) myotubes to either tuncamycine or palmitate induced ER stress and altered insulin-stimulated PKB phosphorylation. However, alleviation of ER stress by either TUDCA or 4-PBA treatments, or by overexpressing Grp78, did not restore palmitate-induced reduction of insulin-stimulated PKB phosphorylation in C(2)C(12) myotubes. This work highlights that, even ER stress is associated with palmitate-induced alterations of insulin signaling, ER stress is likely not the major culprit of this effect in myotubes, suggesting that the previously proposed link between ER stress and insulin resistance is less important in skeletal muscle than in adipose tissue and liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants that exert adverse effects on reproductive processes. In testis, Leydig cells which produce testosterone are under hormonal and local control exerted by cytokines including TNFα. Using mouse Leydig primary cell cultures as a model, we studied the effects of TCDD on the steroidogenic outcome of Leydig cells and the gene expression levels of Ccl5 and Cxcl4, previously shown to be target genes of TCDD in testis. We found that TCDD did not alter the steroidogenic outcome of Leydig cells but that it up-regulated Cxcl4 gene expression levels. TCDD also impacted Ccl5 gene expression when cells had been co-treated with TNFα. TCDD action probably initiated with binding to the aryl hydrocarbon receptor (AhR) present on Leydig cells. TCDD regulated the gene expression levels of AhR (transient down-regulation) and its repressor AhRR and Cyp1b1 (up-regulation). The trophic human chorionic gonadotropin (hCG) hormone did not impact AhR, its repressor AhRR or Cyp1b1 but it opposed the TCDD-enhanced AhRR mRNA levels. Conversely, TNFα stimulated AhR gene expression levels. Collectively, it is suggested that the impact of TCDD on expression of target genes in Leydig cells may operate under the complex network of hormones and cytokines.

International audience; Imeglimin is the first in a new class of oral glucose-lowering agents currently in phase 2b development. Although imeglimin improves insulin sensitivity in humans, the molecular mechanisms are unknown. This study used a model of 16-week high-fat, high-sucrose diet (HFHSD) mice to characterize its antidiabetic effects. Six-week imeglimin treatment significantly decreased glycemia, restored normal glucose tolerance, and improved insulin sensitivity without modifying organs, body weights, and food intake. This was associated with an increase in insulin-stimulated protein kinase B phosphorylation in the liver and muscle. In liver mitochondria, imeglimin redirects substrate flows in favor of complex II, as illustrated by increased respiration with succinate and by the restoration of respiration with glutamate/malate back to control levels. In addition, imeglimin inhibits complex I and restores complex III activities, suggesting an increase in fatty acid oxidation, which is supported by an increase in hepatic 3-hydroxyacetyl-CoA dehydrogenase activity and acylcarnitine profile and the reduction of liver steatosis. Imeglimin also reduces reactive oxygen species production and increases mitochondrial DNA. Finally, imeglimin effects on mitochondrial phospholipid composition could participate in the benefit of imeglimin on mitochondrial function. In conclusion, imeglimin normalizes glucose tolerance and insulin sensitivity by preserving mitochondrial function from oxidative stress and favoring lipid oxidation in liver of HFHSD mice.

International audience; A growing body of evidence suggests that exposure to traffic-related air pollution is a risk factor for type 2 diabetes. Ozone, a major photochemical pollutant in urban areas, is negatively associated with fasting glucose and insulin levels, but most aspects of this association remain to be elucidated. Using an environmentally realistic concentration (0.8 parts per million), we demonstrated that exposure of rats to ozone induced whole-body insulin resistance and oxidative stress, with associated endoplasmic reticulum (ER) stress, c-Jun N-terminal kinase (JNK) activation, and disruption of insulin signaling in skeletal muscle. Bronchoalveolar lavage fluids from ozone-treated rats reproduced this effect in C2C12 myotubes, suggesting that toxic lung mediators were responsible for the phenotype. Pretreatment with the chemical chaperone 4-phenylbutyric acid, the JNK inhibitor SP600125, or the antioxidant N-acetylcysteine alleviated insulin resistance, demonstrating that ozone sequentially triggered oxidative stress, ER stress, and JNK activation to impair insulin signaling in muscle. This study is the first to report that ozone plays a causative role in the development of insulin resistance, suggesting that it could boost the development of diabetes. We therefore provide a potential mechanism linking pollutant exposure and the increased incidence of metabolic diseases.

International audience; Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D). However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFalpha expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.

Metalloproteases (MMPs) are likely to be involved in the restructuring events occurring in the testis throughout development. We here demonstrate that membrane-type 1 (MT1)-MMP, a physiological activator of proMMP-2 under TIMP-2 control, is present within the testis together with MMP-2 and TIMP-2. In the prepubertal testis MT1-MMP immunoreactivity was uniformly distributed, whereas in the adult it was confined to the apical compartment of the tubules, where meiosis and spermiogenesis occur. We further showed that the two cell lineages (somatic and germinal) expressed MT1-MMP and TIMP-2, whereas MMP-2 was of somatic origin. To get a better picture into proMMP-2 activation, use was made of a model of cultured Sertoli cells treated with FSH or co-cultured with germ cells to mimic an immature or a mature developmental period, respectively. We found that follicle-stimulating hormone enhanced the expression of MMP-2 and TIMP-2 but not of MT1-MMP, and promoted the activation of proMMP-2. In co-cultures, a tremendous elevation and activation of MMP-2 was observed, which might relate to the processed MT1-MMP form solely detected in germ cells. That MMP-2 synthesis and activation are under local (germ cells) and hormonal (follicle-stimulating hormone) regulation emphasizes the importance of MMPs in testicular physiology.

The aim of the present study was to identify the sites of the inhibitory action of TNFalpha (tumor necrosis factor alpha) on LH/hCG-stimulated testosterone formation. By using cultured porcine Leydig cells as a model, TNFalpha was shown to inhibit testosterone secretion when testicular cells were stimulated with hCG but not when incubated with 22R-hydroxycholesterol (a cholesterol substrate derivative that readily passes through cell and mitochondrial membranes). Such an observation suggested that the cytokine may affect cholesterol transport and/or availability to cytochrome P450scc in the mitochondria. Specifically, we report here that TNFalpha reduced in a dose- and time-dependent manner hCG-induced StAR (steroidogenic acute regulatory protein) levels. The maximal and half-maximal effects were obtained with 20 ng/ml (1.2 nM) and 1.6 ng/ml (0.09 nM) of TNFalpha, respectively. Maximal inhibitory effects of TNFalpha on StAR messenger RNA and protein levels were obtained after 48 h of treatment. Additionally, the presence of TNFalpha receptors P55 in terms of protein (identified through cross-linking experiments) and messenger RNA (identified through RT-PCR analysis) suggested that the effects of the cytokine are directly exerted on the testicular steroidogenic cell type.

As shown in a large clinical prospective trial, inhibition of the renin-angiotensin system (RAS) can delay the onset of type 2 diabetes in high-risk individuals. We evaluated the beneficial effects of RAS inhibition on β-cell function under glucotoxic conditions. Human islets from 13 donors were cultured in 5.5 mM (controls) or 16.7 mM glucose [high glucose (HG)] for 4 d with or without losartan (5 μM), a selective AT1R blocker, and/or U73122 (2 μM), a selective PLC inhibitor, during the last 2 d. HG induced RAS activation with overexpression of AT1R (P

International audience; There is a growing interest in the optimization of dietary emulsions for monitoring postprandial lipid metabolism in the frame of preventing metabolic diseases. Using various emulsions, we investigated in a systematic scheme the combination of (i) in vitro gastrointestinal lipolysis and (ii) absorption and metabolism of lipolysis media in Caco-2 cells. Four emulsions based on either milk fat olein (OL) or rapeseed oil (RA) as the dispersed phase and either lecithin (LE) or sodium caseinate (CA) as the emulsifier were tested. After a sequential incubation of these emulsions with gastric and pancreatic enzymes, lipolysis media were incubated with Caco-2 cells, after dilution (1 : 20) to maintain the barrier integrity. Both gastric and duodenal lipolysis levels were similar to values reported in vivo and the rates of lipolysis were higher with LE-stabilized emulsions than with CA-stabilized emulsions (P < 0.05). TAG secretion by Caco-2 cells was found to be higher using (i) duodenal vs. gastric media (P < 0.001) and (ii) emulsions stabilized with CA vs. LE (P < 0.01). Consistently, gene expression of both FABP2 and FATP4 induced by the duodenal media was (i) higher than that with gastric media (P < 0.001) and (ii) faster than that with model mixed micelles. Using gastric media, TAG secretion of Caco-2 cells after 12 h was higher with RA than with OL (P < 0.001). Moreover, the RA-CA emulsion increased the size of secreted lipoprotein particles (514 nm vs. 61 to 130 nm; P < 0.01). In conclusion, it was possible to observe distinct responses in the lipid metabolism of Caco-2 cells incubated with lipolysis media obtained from different dietary emulsions digested by gastrointestinal lipases in vitro.

Using as a model system, primary cultures of porcine Leydig cells, we have shown that transforming growth factor-beta 1 (TGF-β1) (2 ng/ml, 72 h) antagonizes the stimulatory action of insulin-like growth factor-I (IGF-I) on luteinizing hormone (LH/hCG) receptors. We therefore investigated the action of TGF-β1 on the different components of the IGF system, namely, IGF-I, II, IGF binding proteins (IGFBPs) and IGF-I receptor present in testicular Leydig cells. TGF-β1 was shown to decrease in a dose and time dependent manner the binding of 125I-IGF-I to Leydig cells. The maximal (40% decrease) effect was obtained with 1.3 ng/ml (0.05 nM) after 72 h of treatment. Such a decrease in IGF-I binding by TGF-β1 treatment was shown to be related to the number of receptor but not to their affinity. Affinity labeling of these receptors by covalently binding them to 125I-IGF-I with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complex revealed that the inhibitory action of TGF-β1 (2 ng/ml, 72 h) occurs at the level of a 135 kDa protein which represents the classical form of the binding subunit of the IGF-I receptor. Moreover, our study indicates that TGF-β1 was unable to affect the other components of the IGF system in cultured porcine Leydig cells. Indeed, TGF-β1 (2 ng/ml, 72 h) was without effect on immunoreactive IGF-I and IGF-II secretion and also on the different IGF binding proteins (IGFBPs) (44, 40, 34, 29, and 24 kDa) as evaluated by ligand blotting analysis. Together, our findings clearly suggest that in Leydig cells TGF-β1 reduces IGF-I action probably by decreasing IGF-I receptor levels but not by affecting the other components of the IGF system. Such a decrease in IGF-I receptors may provide an explanation for the antagonistic effect of TGF-β1 on IGF-I action in Leydig cells.

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H2O2 and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H2O2 production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.

The effect and mechanism of action of basic fibroblast growth factor (bFGF) on testicular steroidogenesis were investigated using as a model primary cultures of purified porcine Leydig cells from immature intact animals. Basic FGF increased basal and human chorionic gonadotrophin (hCG)-induced testosterone accumulation (with an ED50 of 0.64 ng/ml bFGF, 35 pM) in the medium following a long-term treatment. The effects of bFGF (10 ng/ml, 72 h) were found at all hCG concentrations tested (0.001–1 ng/ml), the growth factor affecting the maximal steroidogenic capacity of the Leydig cells but not their sensitivity to the gonadotrophin. In this context, we have therefore investigated whether the stimulatory effect of bFGF on testosterone formation was related to an increase of the steroidogenic enzyme activities. The data obtained indicate that the growth factor did not affect the gonadotrophin action on the formation of Δ 5 - steroid hormone, namely dehydroepiandrosterone (DHEA) (evaluated in the presence of 10−5 M WIN 24540, an inhibitor of 3β-hydroxysteroid dehydrogenase/isomerase). By contrast, bFGF (10 ng/ml, 72 h) was found to increase in a comparable manner the conversion of pregnenolone, DHEA and Δ 4 - androstenedione into testosterone, suggesting a stimulatory effect on 17β-hydroxysteroid dehydrogenase activity. Indeed, bFGF enhanced in a dose-dependent manner ( ED 50 = 39 pM ) this enzyme activity evaluated through the conversion of Δ 4 - androstenedione to testosterone. These effects of bFGF on Leydig cell steroidogenic activity are probably exerted through specific membrane bFGF receptors. Scatchard analysis of the binding of bFGF to cultured purified Leydig cells revealed the presence of a high affinity ( K d = 35.5 pM ) binding site system for bFGF. Affinity labelling to these receptors by covalent attachment to 125I-FGF with disuccinimidyl suberate and subsequent electrophoretic analysis of the labelled complexes revealed the specific binding to two predominant molecules of 145 and 125 kDa. In conclusion, the present study demonstrates that bFGF enhanced steroidogenesis probably via an increase of 17β-hydroxysteroid dehydrogenase activity. Such an effect is likely exerted directly on testicular Leydig cells as shown by the presence of specific membrane bFGF receptors on these testicular cells in primary cultures.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, we have tested the direct effects of tumor necrosis factor-alpha (TNF-alpha) on basal and human (h)CG-stimulated testosterone secretion by cultured purified Leydig cells isolated from immature porcine testes. TNF-alpha reduced (as much as 90% decrease) hCG-stimulated, but not basal testosterone secretion in a dose- and time-dependent manner. The maximal and half-maximal effects were, respectively, 3.75 ng/ml (2.2 x 10(-10) M) and 0.66 ng/ml (3.9 x 10(-11) M) of TNF-alpha after 48 h treatment. TNF-alpha antagonizes the gonadotropin hormonal action by affecting at least two types of biochemical steps. First, TNF-alpha reduced LH/hCG binding to a maximal decrease of 45% obtained with 2 ng/ml of TNF-alpha after 48 h of treatment. TNF-alpha also inhibited (44% decrease) hCG-stimulated cAMP production in optimal conditions (20 ng/ml, 72 h). Second, TNF-alpha significantly (P less than 0.001) reduced testosterone secretion stimulated with 8-bromo-cAMP (3 x 10(-3) M) in a similar range (86% decrease) to that observed with the gonadotropin. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step(s) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 micrograms/ml, 2 h) reversed most of the inhibitory effect of TNF-alpha on androgen production. Indeed, the TNF-alpha (20 ng/ml, 72 h) inhibitory effect on testosterone production was limited to about 20% (P less than 0.03) in Leydig cells supplied with 22R-hydroxycholesterol. Such a moderate effect of the cytokine in the presence of 22R-hydroxycholesterol compared with that observed when androgen secretion was stimulated with the gonadotropin (up to 90% inhibition) indicate that TNF-alpha acts by dramatically reducing cholesterol substrate availability in the mitochondria. Such an effect of TNF-alpha is directly exerted on Leydig cells since TNF-alpha receptors (dissociation constant approximately 5.4 x 10(-10) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells TNF-alpha antagonizes the gonadotropin action on testosterone formation predominantly through a decrease in the availability of cholesterol substrate in the mitochondria.

In the present study, we evaluated the effect of the homodimer activin A on immature porcine Leydig cell functions in primary culture. Activin A (0.5-100 ng/ml) reduced hCG-stimulated dehydroepiandrosterone (DHEA) accumulation in a dose- and time-dependent manner, with a maximal inhibitory effect (58% decrease) at 20 ng/ml (8 x 10(-10) M). Activin A was found not to control steroidogenesis, either through a modulation of the gonadotropin LH/hCG binding or low-density lipoprotein cholesterol binding and internalization. However, activin A significantly decreased pregnenolone (p less than 0.002) and DHEA (p less than 0.001) formation (evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase [3 beta-HSDI]activity) in Leydig cells maximally stimulated with hCG (3 ng/ml, 3 h) or incubated in the presence of 22R-hydroxycholesterol (5 micrograms/ml, 2 h). These findings indicate that activin A probably exerts a partial inhibitory effect on cholesterol side-chain cleavage cytochrome P450 (P450scc) activity. On the other hand, activin A significantly (p less than 0.001) enhanced the conversion of exogenous pregnenolone and DHEA (500 ng/ml) but not of progesterone and androstenedione (500 ng/ml) into testosterone, suggesting that activin A potentially enhances 3 beta-HSDI activity in Leydig cells. Activin A action on 3 beta-HSDI activity was found to be closely related to that of transforming growth factor-beta 1 (TGF beta 1), since both activin A (20 ng/ml) and TGF beta 1 (2 ng/ml) induced a comparable and non-additive increase in 3 beta-HSDI activity.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; Extracellular matrix (ECM) proteins have been shown to alter Leydig cell steroidogenesis in vitro, substantiating the hypothesis that Leydig cell steroidogenic activity and matrix environment are interdependent events. However, the nature of the ECM components synthesized by Leydig cells and their regulation by LH/human chorionic gonadotropin (hCG) remain unknown. Here, we examine the occurrence of the 11 laminin subunits and the 6 alpha chains of collagen IV (COL4A1-6) by RT-PCR in Leydig cells cultured with or without LH/hCG. Leydig cells were a tumor Leydig cell line (mLTC-1) or 8-week-old mice Leydig cells. Based on PCR data, it is suggested that normal Leydig cells may synthesize a maximum of 11 laminin heterotrimers and the 6 alpha chains of collagen IV. They also may synthesize various proteases and inhibitors of the metzincin family. The mLTC-1 cells have a limited repertoire as compared with normal Leydig cells. Interestingly, none of the ten proteases and inhibitors monitored is under LH-hCG regulation whereas every protease and inhibitor of the serine protease family yet identified in Leydig cells is under gonadotropin regulation. In addition, a few laminin and collagen subunit genes are regulated by LH/hCG. These are laminins alpha3 and gamma3 (Lama3 and Lamc3), Col4a3, and Col4a6, which are negatively regulated by LH/hCG in both Leydig cell types, and Col4a4, which was downregulated in primary cultures but not in mLTC-1 cells. Collectively, the present study suggests that Leydig cells modulate in a selective fashion their matrix environment in response to their trophic hormone. This may alter the steroidogenic outcome of Leydig cells.

International audience; Mitochondrial dysfunction in skeletal muscle has been implicated in the development of type 2 diabetes. However, whether these changes are a cause or a consequence of insulin resistance is not clear. We investigated the structure and function of muscle mitochondria during the development of insulin resistance and progression to diabetes in mice fed a high-fat, high-sucrose diet. Although 1 month of high-fat, high-sucrose diet feeding was sufficient to induce glucose intolerance, mice showed no evidence of mitochondrial dysfunction at this stage. However, an extended diet intervention induced a diabetic state in which we observed altered mitochondrial biogenesis, structure, and function in muscle tissue. We assessed the role of oxidative stress in the development of these mitochondrial abnormalities and found that diet-induced diabetic mice had an increase in ROS production in skeletal muscle. In addition, ROS production was associated with mitochondrial alterations in the muscle of hyperglycemic streptozotocin-treated mice, and normalization of glycemia or antioxidant treatment decreased muscle ROS production and restored mitochondrial integrity. Glucose- or lipid-induced ROS production resulted in mitochondrial alterations in muscle cells in vitro, and these effects were blocked by antioxidant treatment. These data suggest that mitochondrial alterations do not precede the onset of insulin resistance and result from increased ROS production in muscle in diet-induced diabetic mice.

Introduction Les mitochondries ont recemment emerge comme un regulateur du metabolisme energetique lors des transitions nutritionnelles, et leurs interactions avec le RE (zones appelees MAM, mitochondria-associated endoplasmic reticulum membranes) comme un regulateur de l'action de l'insuline dans le foie. Neanmoins, la regulation nutritionnelle des MAM dans le foie et son role dans l'adaptation de la physiologie des mitochondries a la disponibilite en nutriments sont inconnus. Materiels et methodes L'integrite des MAM a ete etudiee, in vivo chez la souris et in vitro dans des cellules HuH7/hepatocytes primaires murins, par fractionnement cellulaire et in situ proximity ligation assay, la dynamique mitochondriale par immunofluorenscence et la respiration par oxygraphie. Resultats La periode post-prandiale reduit l'integrite des MAM dans le foie des souris. L'analyse in vitro des metabolites et/ou signaux hormonaux responsables de cette regulation a revele que le glucose est le principal regulateur nutritionnel de l'integrite des MAM dans les hepatocytes. Une augmentation des concentrations en glucose reduit les interactions inter-organites via la voie des pentoses phosphate (PP) et l'activation de la proteine PP2A, induit une fission des mitochondries et altere la respiration mitochondriale dans les hepatocytes. La prevention de la reduction de MAM, par le blocage de la voie PP-PP2A ou en surexprimant la chaperone GRP75, previent les modifications mitochondriales induites par le glucose. De maniere interessante, ce « sensing » du glucose est deficient dans le foie de souris ob/ob, caracterise par une alteration de l'integrite des MAM, une fission et dysfonction mitochondriales et une mauvaise adaptation aux transitions nutritionnelles. Conclusion Les MAM constituent un nouveau systeme de detection du glucose hepatique permettant la regulation de la dynamique et la fonction des mitochondries au cours des transitions nutritionnelles. Leurs deregulations dans le foie des souris ob/ob suggerent l'implication potentielle des MAM dans la dysfonction mitochondriale associee a l'insulinoresistance hepatique. Declaration d’interet Les auteurs declarent ne pas avoir d'interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

Introduction Une augmentation des acides gras libres plasmatiques est associee a l'obesite et au diabete de type 2, et participe au developpement de l'insulino-resistance. Plus particulierement, les acides gras satures (ex : le palmitate) sont connus pour avoir des effets deleteres via l'induction d'un stress du reticulum endoplasmique (RE) dans de nombreux types cellulaires. Neanmoins, on ne sait pas a l'heure actuelle si les effets du palmitate sur la sensibilite a l'insuline impliquent un stress du RE dans le muscle squelettique. Materiels et methodes Nous avons etudie les repercussions d'un traitement au palmitate sur l'homeostasie du RE et sur la sensibilite a l'insuline, a la fois in vitro , dans des C2C12, et in vivo , dans le muscle de souris soumises a une diete enrichie en huile de palme (souris HPD). L'implication du stress du RE dans l'insulino-resistance induite par le palmitate a ete determinee en traitant des myotubes soit avec du TUDCA (tauroursodeoxycholic acid) soit en surexprimant la chaperone Grp78 dans les C2C12. Resultats Nous demontrons que le palmitate (200–700 μM, 3–48 heures) induit un stress du RE et altere la signalisation de l'insuline (inhibition de la phosphorylation d'Akt induite par l'insuline) dans les myotubes differencies. L'inhibition du stress du RE, soit par un traitement au TUDCA soit par la surexpression de la proteine chaperone Grp78, n'ameliore pas l'insulino-resistance induite par le palmitate. L'induction d'un stress du RE par le palmitate est confirmee in vivo dans le muscle gastrocnemius de souris HPD, alors que la sensibilite a l'insuline du muscle n'est pas encore alteree apres 8 semaines de traitement. Conclusion L'ensemble de ces donnees demontrent que l'induction du stress du RE par le palmitate n'est pas impliquee dans le developpement de l'insulino-resistance musculaire.

Introduction La perturbation de l'homeostasie calcique en conditions de lipotoxicite entraine une dysfonction beta-pancreatique. Une etude recente a montre que l'ouverture du translocon, complexe multiproteique implique dans la traduction, entraine une fuite calcique reticulaire passive. Nous etudions ici le role du translocon sur la fonction beta lors d'un traitement au palmitate. Materiels et methodes Les [Ca 2+ ] cyt et [Ca 2+ ] RE des ilots humains et des MIN6B1 ont ete mesurees avec la sonde Fura-2 AM. La presence fonctionnelle du translocon a ete determinee par la mesure du [Ca 2+ ] RE apres ajout d'anisomycine (200μM) et/ou de puromycine (200μM), respectivement inhibiteur et ouvreur du translocon. Les MIN6B1 ont ete cultivees en presence de BSA ou de palmitate (200μM) et d'anisomycine (200 nM) durant 24h. La secretion d'insuline en reponse au glucose (GSIS) a ete determinee par ELISA ; les marqueurs du stress du RE, la proteine majeure du translocon SEC61a, par qPCR et western-blot. Resultats La puromycine entraine une fuite reticulaire dans les ilots humains (p=0,05) et les MIN6B1 (p 2+ ] RE dans les MIN6B1 (p Conclusion Nous demontrons pour la premiere fois que le translocon est implique dans l'homeostasie calcique des cellules beta et que son inhibition protege leur fonction en conditions de lipotoxicite. Le translocon apparait ainsi comme une nouvelle piste therapeutique potentielle dans le diabete de type 2.

Introduction La lipotoxocite chronique lies aux acides gras libres entraine une apoptose beta pancreatiques et peut conduire au diabete de type 2 (DT2). L’inhibition du systeme renine-angiotensine (SRA) a ete montree comme preventive du DT2 chez des patients a risque. Nous faisons l’hypothese que ces effets protecteurs passent par une action directe sur les cellules beta, un SRA local ayant ete demontre. Nous avons analyse les effets du Losartan, sur les fonctions reticulaire et mitochondriale, ainsi que sur l’homeostasie calcique des cellules beta murines exposes a une dose chronique de palmitate. Materiels et methodes Les cellules MIN6B1 ont ete cultives en presence de BSA ou de palmitate (200 μM) durant 24 h, avec ou sans Losartan (5 μM). Resultats Le palmitate active le SRA en induisant une augmentation de l’angiotensinogene et l’ACE (p Conclusion Le Losartan protege les cellules Beta de la lipotoxicite induite par le palmitate en normalisant l’homeostasie calcique et les fonctions reticulaire et mitochondriale. La mise en jeu du SRA dans ces regulations suggere son role majeur dans la cellule beta. Ce qui pourrait ouvrir de nouvelles perspectives therapeutiques dans la prevention du DT2.

Introduction L’environnement lipidique joue un role important dans les alterations fonctionnelles des cellules β, principalement en alterant les mitochondries et le reticulum endoplasmique (RE). Neanmoins, les mecanismes a l’origine des alterations de ces 2 organites sont encore mal connus. L’objectif de notre etude etait d’analyser in vivo et in vitro les effets du palmitate sur l’integrite des mitochondries et du RE des cellules β, et de determiner si leurs alterations en situation de lipotoxicite etaient inter-reliees. Materiels et methodes Des cellules MIN6B1 ont ete incubees avec du palmitate (200 μM, 24 h). Des ilots β ont ete isoles de pancreas de souris nourries soit avec un regime standard soit un regime enrichi en huile de palme (HPD, 20 %, 8 semaines). Nous avons mesure les repercussions sur la respiration mitochondriale, le stress du RE, les interactions mitochondrie/RE et la secretion d’insuline. Les interactions mitochondrie/RE ont ete egalement etudiees (in situ PLA) dans des ilots de souris ob/ob et high-fat-diet16sem. Resultats Le traitement des cellules MIN6B1 par le palmitate (200 μM) induit un stress du RE, altere la respiration mitochondriale, diminue les interactions mitochondrie/RE et inhibe la secretion d’insuline induite par le glucose. Un co-traitement au 4-Phenyl-Butyric Acid (4-PBA) ameliore la respiration mitochondriale sans restaurer significativement les interactions mitochondrie/RE et la secretion d’insuline. Les ilots pancreatiques des souris HPD n’augmentent plus leur secretion d’insuline en reponse au glucose et presentent un defaut de respiration mitochondriale, associe a une alteration des interactions mitochondrie/RE, qui ne sont pas prevenus par le 4-PBA. Enfin, nous confirmons une alteration des interactions inter-organelles dans les ilots de souris ob/ob et HFD16 sem. Conclusion Nos resultats demontrent un effet deletere du palmitate sur la fonctionnalite des cellules β qui pourrait impliquer un defaut d’interaction entre la mitochondrie et le RE en situation de lipotoxicite, qui n’est pas prevenu par le 4-PBA.

Introduction L’insulino-resistance hepatique (IRH) est associee a une dysfonction mitochondriale et a un stress du reticulum endoplasmique (RE). Neanmoins, on ne sait pas a l’heure actuelle si ces alterations sont interreliees ou representent des facteurs independants. De maniere interessante, ces deux organites sont intimement lies via des points de contacts, appeles Mitochondria Associated Membranes (MAM), qui jouent un role primordial dans la signalisation du calcium et le metabolisme energetique. Les transferts calciques du RE vers la mitochondrie impliquent le complexe proteique VDAC1/Grp75/IP3R1. Le but de notre etude etait de determiner si la diminution des interactions dans ce complexe pouvait jouer un role causal dans l’induction du stress du RE et de l’IRH. Materiels et methodes Nous avons estime la quantite de MAM par fractionnement cellulaire dans le foie de souris ob/ob, un modele d’IRH et de stress du RE. De plus, nous avons mesure les repercussions de l’inhibition pharmacologique et/ou genique du complexe VDAC1/Grp75/IP3R1 sur l’interaction des organites (par in situ proximity ligation assay), le stress du RE et sur la signalisation de l’insuline dans les cellules HuH7. Resultats Nous demontrons dans le foie des souris ob/ob une diminution de la quantite de MAM (– 50 %, p Conclusion Nos resultats demontrent un nouveau role des interactions mitochondrie/RE dans le developpement du stress du RE et l’IRH. Ils suggerent aussi un role d’une alteration de l’homeostasie calcique comme lien mecanistique entre ces 2 phenomenes.

Introduction S’il est bien admis que l’insulino-resistance hepatique (IRH) est associee a des alterations mitochondriales, un stress du reticulum endoplasmique (RE) et une perturbation de l’homeostasie calcique, on ne sait pas a l’heure actuelle si ces defauts representent des facteurs independants. Nous avons teste l’hypothese qu’ils sont inter-relies, via une alteration des echanges calciques au niveaux des points de contact entre les deux oragnites, appeles mitochondria-associated membranes (MAM). Materiels et methodes Sur la base de donnees recentes impliquant une chaperone mitochondriale, la cyclophiline D, dans la regulation de l’homeostasie calcique, du RE et du metabolisme glucidique, nous avons utilise des souris invalidees pour la cypD (cypD-KO) et des approches pharmacologiques et geniques in vitro pour etudier le role de la signalisation calcique mediee par IP3R au niveau des MAM dans l’IRH. Resultats Les souris cypD-KO presentent un stress du RE hepatique responsable d’une steatose, d’une IRH et d’une perturbation de l’homeostasie glucidique, puisque ces alterations sont prevenues par une inhibition du stress du RE (traitement au TUDCA ou surexpression hepatique de la chaperone Grp78). Ce stress du RE est lie a un defaut d’interaction et d’echange calcique entre le RE et la mitochondrie dans les hepatocytes de souris cypD-KO. L’ensemble de ces donnees sont reproduites in vitro dans des cellules HuH7 ou la cypD a ete inhibee soit par des traitements pharmacologiques (cyclosporine et NIM811) soit par un ARNi specifique. Enfin, nous demontrons que les alterations de la signalisation de l’insuline induite par la perte de cypD sont mediees par une activation de la PKCɛepsi; plutot que de la JNK. Conclusion Nos resultats demontrent un nouveau role de la cypD dans les interactions structurales et fonctionnelles entre le RE et la mitochondrie et un role cle de la signalisation calcique mediee par IP3R au niveau des MAM dans l’insulino-resistance hepatique.

Introduction Nous avions montre que l’inhibition du systeme renine-angiotensine (SRA) beta-pancreatique par le Losartan, inhibiteur de l’AT1R, protege les cellules Beta du stress du RE induit par le glucose et restaure la secretion d’insuline. L’activation du SRA active la voie PLC-IP3-Calcium. Nous avons analyse les effets du Losartan et d’un inhibiteur de la PLC (U73122) sur la fonction mitochondriale et l’homeostasie calcique d’ilots pancreatiques humains exposes a une dose elevee de glucose (HG). Materiels et methodes Les ilots humains de 11 donneurs ont ete cultives en presence de 5,5 mM ou 16,7 mM de glucose durant 96 h, avec ou sans Losartan (5 μM) ou U73122 (2 μM) les dernieres 48 h. Resultats Nous avions montre dans les ilots humains une production des especes reactives de l’oxygene (ERO) accrue en milieu HG et reduite par le Losartan. L’angiotensine II augmente la production d’ERO via la voie de la NADPH oxidase dans des ilots de rat. Nous montrons que l’augmentation de l’angiotensinogene (p Conclusion Le Losartan abroge les effets deleteres du glucose sur la fonction mitochondriale et l’homeostasie calcique des ilots pancreatiques humains. Ces resultats suggerent que le SRA joue un role majeur dans la physiologie de la cellule beta et que son inhibition pourrait jouer un role dans la prevention du diabete de type 2.

Introduction Le gene FTO (fat mass and obesity-associated), fortement associe a l’obesite et au diabete de type 2 dans de nombreuses cohortes, a ete depuis implique dans le controle de la prise alimentaire via STAT3. Dans le foie, l’expression de FTO a ete correlee positivement avec l’expression des genes de la neoglucogenese, et STAT3 est un acteur important au controle de la neoglucogenese hepatique. L’objectif de notre etude etait donc d’evaluer le role de FTO dans le controle de la neoglucogenese hepatique et l’implication de la voie STAT3. Materiels et methodes Nous avons surexprime FTO in vivo dans le foie de souris par infection adenovirale et in vitro dans des cellules HuH7 par transfection transitoire, et etudie les repercussions sur la neoglucogenese et la regulation de la signalisation via STAT3. Resultats Les souris surexprimant FTO (Ad-FTO) specifiquement dans le foie ont une glycemie, une insulinemie et une leptinemie a jeun augmentees. Des tests de tolerance au glucose montrent que ces souris sont intolerantes au glucose. L’expression de genes impliques dans la neoglucogenese (G6P, FOXO1a) est augmentee, alors que l’expression de genes impliques dans la signalisation de la voie leptine/STAT3 (LepR, SOCS3) est diminuee. De plus, la forme phosphorylee de STAT3 est diminuee dans le noyau des souris Ad-FTO. In vitro, la surexpression de FTO dans des HuH7 exprimant le recepteur de la leptine induit des alterations de la reponse a la leptine au niveau de la phosphorylation de STAT3, entrainant une diminution de l’expression de SOCS3 et a une augmentation de la G6P. Conclusion La surexpression hepatique de FTO in vivo et in vitro affecte la signalisation STAT3 regulee par la leptine et induit les genes de la neoglucogenese, suggerant un role important de FTO dans le controle de la neoglucogenese hepatique via la voie STAT3.

Introduction Le diabete type 2 (DT2) est associe a une inflammation a bas bruit et une accumulation de macrophages pro-inflammatoires principalement dans le tissu adipeux. Cette inflammation participerait au developpement de l’insulino-resistance (IR), puisque les cytokines pro-inflammatoires inhibent la signalisation de l’insuline. Le muscle squelettique est le principal tissu utilisateur de glucose en reponse a l’insuline, et une cible privilegiee des alterations en situation d’IR. Neanmoins, il existe tres peu d’informations sur le statut inflammatoire du muscle dans le cadre du DT2. L’objectif de notre etude a ete d’etudier l’infiltration de macrophages dans le muscle squelettique de differents modeles de souris diabetiques et le role des lipides dans l’attraction des macrophages. Materiels et methodes Nous avons mesure l’infiltration de macrophages (immunohistochimie) et l’expression de cytokines pro- et anti-inflammatoires (PCR en temps reel) dans le muscle gastrocnemius de souris ob/ob et de souris soumises soit a une diete riche en lipides (HFD 45 %) soit a des dietes enrichies en differents types d’acide gras (tournesol, colza, beurre ou palme, 20 %). L’effet benefique d’un traitement antidiabetique (Rosiglitazone) a egalement ete etudie chez les souris HFD. L’implication des lipides dans l’attraction des macrophages a ete etudie in vitro en exposant des macrophages Raw264.7 a des milieux conditionnes (MC) de C2C12 incubes ou pas en presence de palmitate (700 μm). Resultats Nous observons une infiltration de macrophages dans le muscle gastrocnemius des differents modeles de souris diabetiques, qui est positivement correlee a l’expression des cytokines pro-inflammatoires (TNFa, IL1b) et a IR. Le recrutement des macrophages semble faire intervenir la secretion de chemokines (MCP1) par les cellules musculaires, puisque l’exposition des Raw264.7 a des MC de C2C12 exposes au palmitate augmente leur migration (p Conclusion Nos resultats demontrent que le DT2 s’accompagne d’une infiltration de macrophages pro-inflammatoires dans le muscle squelettique, qui pourrait participer a l’installation de l’IR.

The present study examines how the hormonal action of gonadotropin is modulated by transforming growth factor-beta 1 (TGF beta 1) and epidermal growth factor (EGF) in primary cultures of purified porcine Leydig cells. Although TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) individually enhanced hCG-stimulated testosterone formation, the effects of EGF were more pronounced than those of TGF beta 1. When studied in combination, the effects of maximal concentrations of TGF beta 1 and EGF were additive on gonadotropin hormonal action. In the present study we demonstrate that their additive effects resulted from a complex interaction occurring at the levels of cholesterol substrate availability in the mitochondria and of 3 beta-hydroxysteroid dehydrogenase/isomerase activity (3 beta HSDI). First, TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) were, respectively, shown to reduce and enhance dehydroepiandrosterone (DHEA) formation (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta HSDI) in Leydig cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml), but not when incubated with 22R-hydroxycholesterol (3 micrograms/ml). Such findings indicate that TGF beta 1 and EGF did not affect cholesterol side-chain cleavage cytochrome P450 activity, but, respectively, decreased and increased cholesterol substrate availability for this enzyme in the mitochondria. Furthermore, when Leydig cells were treated with the combined factors, the formation of delta 5-steroid intermediates (such as DHEA) in untreated (control) and EGF-plus TGF beta 1-treated cells was not significantly different whether the cells were acutely stimulated with the gonadotropin or incubated with 22R-hydroxycholesterol. Such findings indicate that the effects of EGF and TGF beta 1 on cholesterol substrate availability in the mitochondria are antagonistic. Second, EGF, TGF beta 1, and EGF plus TGF beta 1 significantly (P less than 0.001) increased delta 5-steroid intermediate (i.e. pregnenolone and DHEA), but not delta 4-steroid intermediate (i.e. progesterone and androstenedione), conversion into testosterone, indicating that the growth factors increased, individually or in combination in an additive manner, 3 beta HSDI activity (respectively, 90.7 +/- 0.6%, 80.6 +/- 2.6%, and 164 +/- 4.5% increase in the presence of EGF, TGF beta 1, and EGF plus TGF beta 1). Together, the reciprocal suppression of the effects of TGF beta 1 and EGF on the mitochondrial cholesterol substrate availability coupled to their stimulatory additive actions on 3 beta HSDI activity provide an explanation of the additive actions of the two growth factors on gonadotropin-induced testicular androgen formation.

In the present study, we have tested the effects of transforming growth factor beta 1 (TGF beta 1) on FSH action toward aromatase activity and lactate production in cultured Sertoli cells isolated from immature porcine testes. Whereas treatment of Sertoli cells with FSH resulted in a dose-dependent increase (about 7-fold) in aromatase activity (conversion of testosterone into estradiol) (ED50 = 80 ng/ml FSH), the addition of TGF beta 1 reduced this gonadotropin action. The inhibitory effect of TGF beta 1 on FSH aromatase activity was dose dependent (ED50 = 0.1 ng/ml, 4 pM TGF beta 1) with a maximal decrease (about 40%) observed after a long term (48-h) treatment. TGF beta 1 exerted its inhibitory effect on FSH action at the level(s) of cAMP accumulation, exerting no apparent effect on the gonadotropin receptor or at a site(s) related to cAMP action. TGF beta 1 (2 ng/ml) significantly (P less than 0.002) reduced (52% decrease) FSH-stimulated cAMP levels in cultured porcine Sertoli cells. However, such an inhibitory effect of the growth factor was no longer observed when stimulation of cAMP accumulation with FSH occurred in the presence of methyl isobutyl xanthine (0.5 mM), an inhibitor of cAMP-phosphodiesterase activity. This observation suggests that TGF beta 1 decreased cAMP levels by increasing catabolism of the cyclic nucleotide through an enhancement of cAMP-phosphodiesterase activity. The inhibitory effect of TGF beta 1 was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action (mediated by cAMP) on lactate production. As for the inhibitory effect of TGF beta 1 on FSH-induced aromatase activity, the inhibitory effect of the growth factor on FSH-stimulated lactate production was dose and time dependent with a maximal decrease (about 30%) observed in the picomolar range (1 ng/ml, 40 pM) after 48 h treatment with TGF beta 1. In conclusion, the present study demonstrates that TGF beta 1 attenuates FSH action on Sertoli cell activity and that such inhibitory action is potentially exerted through a decrease in cAMP levels. Because of the local production of TGF beta 1, it is suggested that the effects of the growth factor reported here might be exerted in the context of the testicular paracrine mechanisms.

Introduction Des dysfonctionnements mitochondriaux dans le muscle squelettique seraient impliques dans le developpement du diabete de type 2. Cependant, si ces alterations sont une cause ou une consequence de l’insulino-resistance n’est toujours pas determine. Materiels et methodes Nous avons donc etudie la structure et les fonctions mitochondriales au cours du developpement de l’insulino-resistance, induite chez l’animal par une diete riche en lipides et en sucrose. Resultats Aucune alteration mitochondriale n’est mis en evidence dans le muscle de souris prediabetiques, alors que la biogenese, la structure et les fonctions mitochondriales sont alterees dans le muscle de souris diabetiques. D’autre part, nous demontrons que le stress oxydant musculaire est un element causal majeur dans ces alterations mitochondriales puisque 1. il existe une augmentation de la production locale de radicaux libres (RL) uniquement dans le muscle squelettique de souris diabetiques, associee a une hyperglycemie et hyperlipidemie, 2. Une production de RL est egalement associee a des alterations mitochondriales dans le muscle de souris de souris rendues hyperglycemiques par un traitement a la streptozotocine, 3. Chez ces souris, la normalisation de la glycemie par un traitement a l’insuline ou a la phlorizine, ainsi qu’un traitement antioxydant, diminuent la production de RL et restaurent les alterations mitochondriales, et 4. l’induction d’une production de RL par du glucose ou des lipides, in vitro, altere la densite et les fonctions mitochondriales dans des cellules musculaires en culture, et ces effets sont restaures par un traitement antioxydant. Conclusion L’ensemble de ces donnees suggerent que les alterations mitochondriales ne sont pas causales dans le developpement de l’insulino-resistance, mais plutot la consequence d’un stress oxydant musculaires associe a l’hyperglycemie et l’hyperlipidemie des souris diabetiques.

The regulating effect of follicle-stimulating hormone (FSH) on Leydig cell function was studied using a model of immature porcine Leydig and Sertoli cells cultured in a hormone supplemented defined medium. FSH pretreatment for 2 days of Leydig cells cultured alone was with no effect. FSH pretreatment of Leydig cells cocultured with Sertoli cells increases Leydig cell activity in an FSH dose-dependent manner with a maximal effect observed at 50 ng/ml porcine FSH (pFSH). Leydig cells cultured for 2 days in conditioned medium (CM) by FSH stimulated (FSH-CM) Sertoli cells, as compared to CM by unstimulated (control) (C-CM) Sertoli cells show an increase of their activity with a maximal effect observed at 50 ng/ml pFSH. Leydig cells cultured in CM as compared to non CM, show a marked development of organelles (smooth endoplasmic reticulum and mitochondria) involved in the steroidogenic activity. The activity of FSH-CM as compared to C-CM on Leydig cell function was non dialyzable and trypsin sensitive. These data suggest that Sertoli cells exert a regulatory action on Leydig cell steroidogenic activity via FSH dependent secreted proteins.

The regulatory effect of fibroblast growth factor (FGF) on testosterone secretion was studied by using a model of immature porcine Leydig cells cultured in serum-free defined medium. FGF enhanced in a dose-dependent manner hCG-stimulated testosterone secretion (ED50 = 11 ng/ml FGF). The stimulatory effect of FGF on testosterone accumulation was time dependent; testosterone increased to a maximal value at 24 h treatment and then dramatically declined to near control value following 48 and 72 h treatment with FGF; such a decline was not related to FGF degradation in culture medium. Although FGF increased maximal secretion of testosterone, it did not affect the human chorionic gonadotrophin (hCG) concentrations required for maximal and half-maximal secretion of testosterone (1 and 0.2 ng/ml hCG, respectively). These effects of FGF are probably exerted in the context of the local control of testicular steroidogenesis.

The effect of transforming growth factor beta on testicular steroidogenesis was studied by using a model of immature porcine Leydig cells cultured in a chemically defined medium. Leydig cells were cultured in the presence of human or porcine purified TGF beta and the following parameters were measured: cell proliferation, LH/hCG binding, and hCG-stimulated steroid hormone productions (DHEA, DHEAS and testosterone). Whereas TGF beta from the two sources had no effect on Leydig cell multiplication, it markedly inhibited LH/hCG-stimulated DHEA and DHEAS in a time- and dose-dependent manner. The maximal inhibitory effect of this peptide on LH/hCG binding (65% decrease), hCG-stimulated DHEA (77% decrease) and DHEAS (92% decrease) productions was observed with 2 ng/ml for 48 h of treatment. In contrast, TGF beta exerted a biphasic effect on hCG-stimulated testosterone production: stimulating (110% increase) until 2 ng/ml and inhibiting (35% decrease) for higher concentrations. [125I]TGF beta was cross-linked to Leydig cells using disuccinimidyl suberate; cells affinity labelled with [125I]TGF beta exhibit a major labelled band of approx 280 kDa, which has the properties expected from a TGF beta receptor. These data demonstrate that TGF beta is a direct potent regulator of Leydig cell steroidogenic function and its effects are probably mediated via a specific receptor.

By using immature porcine Leydig cells cultured in defined medium as a model, transforming growth factor-beta (TGF beta) was shown to exert a dramatic inhibitory effect on their basal and human chorionic gonadotropin (hCG) (or 8-bromo-cyclic AMP) stimulated dehydroepiandrosterone secretion, in the presence or absence of saturating concentrations of exogenous (low density lipoprotein) cholesterol substrate. In contrast, TGF beta exerted both a stimulating and inhibitory effect on testosterone secretion: while hCG-stimulated testosterone secretion was enhanced by low doses of TGF beta (0.06-0.4 ng/ml, 48 h), it was decreased with higher concentrations of TGF beta (2.5-10 ng/ml, 48 h). The data obtained show that the inhibitory action of TGF beta on testicular steroidogenesis was related to a decrease in pregnenolone formation by affecting a step(s) distal to cyclic AMP formation but before cholesterol association with cytochrome P-450 side-chain cleavage. As for the stimulatory effect of TGF beta on testosterone formation, this was mainly related to an increase (about 2-fold) in 3 beta-hydroxysteroid dehydrogenase/isomerase activity (ED50 0.05 ng/ml, 2 X 10(-13) M). The results indicate that the (short-term) steroidogenic stimulatory action of luteinizing hormone (LH)/hCG is antagonized by high concentrations of TGF beta by decreasing pregnenolone formation while it is enhanced by the stimulating action of low concentrations of TGF beta exerted on 3 beta-hydroxy steroid dehydrogenase/isomerase activity.

The role of ACTH-(1–24) on angiotensin II receptors has been studied in bovine adrenal glomerulosa cells in primary culture. Angiotensin II receptors were measured in cells pretreated or not by ACTH-(1–24) on day 4 of culture. ACTH-(1–24) decreased angiotensin II binding sites in a time and a dose-dependent manner. After 24 hours of treatment the minimal effective dose of ACTH-(1–24) was 10−11M and the maximal effect was obtained with 10−8M. Moreover, ACTH-(1–24) 10−8M decreased significantly angiotensin II receptors after 6 hours of treatment. Scatchard plot analysis showed that ACTH-(1–24) treatment did not modify the affinity of angiotensin II receptors (Ka = 0.42 and 0.44 × 109M−1 in control and treated cells respectively) but reduced by about half the number of angiotensin II sites per cell. Like ACTH-(1–24), 8-Bromo-cAMP, forskolin and cholera toxin decreased angiotensin II receptors. Factors such as prolactin, somatostatin, ACTH-(11–24) and dopamine which are bound to adrenal membranes without increasing cAMP production had no effect. In conclusion, these studies in vitro demonstrate for the first time that ACTH decreases angiotensin II receptors by a direct mechanism acting on glomerulosa cells, and they also suggest that this effect could be mediated by cAMP.

Type beta Transforming Growth Factor (TGF beta)-like activity was identified in conditioned medium obtained from immature porcine Sertoli cell-enriched cultures using the following criteria: (i) stimulation of anchorage independent growth of mesenchymal cell lines, (ii) competition with pure human TGF beta in a radioreceptor assay. The secretion of the receptor reactive TGF beta-like material in Sertoli cell conditioned medium is decreased to very low or undetectable levels by Follicle Stimulating Hormone, one of the major hormones involved in the physiological testicular activities. The effects of this factor are probably exerted in the context of the local control of the male gonad functions.