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The implication of αβ and γδ T cells in obesity-associated inflammation and insulin resistance (IR) remains uncertain. Mice lacking γδ T cells show either no difference or a decrease in high-fat diet (HFD)-induced IR, whereas partial depletion in γδ T cells does not protect from HFD-induced IR. αβ T-cell deficiency leads to a decrease in white adipose tissue (WAT) inflammation and IR without weight change, but partial depletion of these cells has not been studied. We previously described a mouse model overexpressing peroxisome proliferator-activated receptor β (PPAR-β) specifically in T cells [transgenic (Tg) T-PPAR-β] that exhibits a partial depletion in αβ T cells and no change in γδ T-cell number. This results in a decreased αβ/γδ T-cell ratio in lymphoid organs. We now show that Tg T-PPAR-β mice are partially protected against HFD-induced weight gain and exhibit decreased IR and liver steatosis independently of animal weight. These mice display an alteration of WAT-depots distribution with an increased epididymal-WAT mass and a decreased subcutaneous WAT mass. Immune cell number is decreased in both WAT-depots, except for γδ T cells, which are increased in epididymal-WAT. Overall, we show that decreasing αβ/γδ T-cell ratio in WAT-depots alters their inflammatory state and mass repartition, which might be involved in improvement of insulin sensitivity.-Le Menn, G., Sibille, B., Murdaca, J., Rousseau, A.-S., Squillace, R., Vergoni, B., Cormont, M., Niot, I., Grimaldi, P. A., Mothe-Satney, I., Neels, J. G. Decrease in αβ/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

Leukotrienes (LTs) are potent proinflammatory mediators, and many important aspects of innate and adaptive immune responses are regulated by LTs. Key members of the LT synthesis pathway are overexpressed in adipose tissue (AT) during obesity, resulting in increased LT levels in this tissue. We observed that several mouse adipocyte cell lines and primary adipocytes from mice and humans both can secrete large amounts of LTs. Furthermore, this production increases with a high-fat diet (HFD) and positively correlates with adipocyte size. LTs produced by adipocytes play an important role in attracting macrophages and T cells in in vitro chemotaxis assays. Mice that are deficient for the enzyme 5-lipoxygenase (5-LO), and therefore lack LTs, exhibit a decrease in HFD-induced AT macrophage and T-cell infiltration and are partially protected from HFD-induced insulin resistance. Similarly, treatment of HFD-fed wild-type mice with the 5-LO inhibitor Zileuton also results in a reduction of AT macrophages and T cells, accompanied by a decrease in insulin resistance. Together, these findings suggest that LTs represent a novel target in the prevention or treatment of obesity-associated inflammation and insulin resistance.

Resume Une caracteristique majeure du vieillissement est la sarcopenie liee a l’âge, a l’origine de consequences negatives en terme de handicap et de morbidite mais faisant aussi le lit de la denutrition et de la vulnerabilite. A cote d’ingesta proteiques insuffisants, des anomalies du metabolisme proteique sont frequentes, avec une plus forte extraction splanchnique des acides amines et une diminution de leur action anabolisante au niveau musculaire, principalement du fait d’une moindre stimulation de la voie de signalisation mTOR. Une augmentation de la proteolyse (via le complexe ubiquitine–proteasome) est impliquee a un moindre degre. Les consequences de ces anomalies sur les apports en acides amines sont, en premier lieu, de ne jamais se situer sous le seuil d’apports de 1 g/kg par jour de proteines. L’administration des proteines en bolus ainsi que le recours a des proteines rapides sont des pistes prometteuses. Enfin, l’administration de quantites supraphysiologiques de certains acides amines, tels la leucine surtout mais aussi la citrulline et l’arginine, permet de stimuler la synthese proteique, principalement via l’activation de la voie mTOR, et represente une alternative therapeutique interessante a comparer aux pistes medicamenteuses.

Summary Background & aims Anorexia is frequent in the malnourished elderly. We studied the effects of age, nutritional status and refeeding on the expression and secretion of the orexigenic peptide ghrelin. Methods Four groups were prospectively enrolled: 11 undernourished elderly (80 ± 6 y, BMI: 17.4 ± 1.9 [Mean ± SD]), nine well-nourished elderly (76 ± 9 y, 23.5 ± 2.0), 10 undernourished young (26 ± 6 y, 15.1 ± 1.9) and 10 well-nourished young (34 ± 8 y, 22.2 ± 2.7). Fasting and postprandial plasma ghrelin and other hormones (every 30 min) were measured at baseline and after a 21-day enteral nutrition in malnourished patients. Gastric ghrelin mRNA levels were measured by RT-PCR at baseline in all subjects. Results Ghrelin was significantly higher in undernourished (2151 ± 871 ng/L) than in well-nourished (943 ± 389 ng/L) adults, whereas there were no differences between undernourished (1544 ± 758 ng/L) and well-nourished (1154 ± 541 ng/L) elderly. Refeeding did not influence ghrelin levels. Gastric ghrelin mRNA levels were similar in all groups. Conclusions There is an absence of malnutrition-induced increase of plasma ghrelin levels in elderly subjects. This feature, post-transcriptional, may be important in the lack of adaptation of elderly subjects to malnutrition.

We hypothesized that α-lipoic acid (α-LA) might interact with the transcriptional control of peroxisome proliferator-activated receptor (PPAR)β in skeletal muscle. Molecular mechanisms were investigated using differentiated C2C12 myotubes treated with α-LA and/or PPARβ agonist GW0742. In vivo studies with 3-mo-old C57Bl6 mice were realized: voluntary wheel running (VWR) training (7 wk), and a 6 wk diet containing (or not) α-LA (0.25% wt/wt). This last condition was combined with (or not) 1 bout of treadmill exercise (18 m/min for 1 h). Using a reporter assay, we demonstrate that α-LA is not an agonist of PPARβ but regulates PPARβ target gene expression through an active PPARβ pathway. GW0742-induced pyruvate dehydrogenase kinase 4 mRNA is potentiated by α-LA. In C2C12, α-LA lowers the activation of the JNK signaling pathway and increases PPARβ mRNA and protein levels (2-fold) to the same extent as with the JNK inhibitor SP600125. Similarly to VWR training effect, PPARβ expression increases (2-fold) in vastus lateralis of animals fed an α-LA-enriched diet. However, α-LA treatment does not further stimulate the adaptive up-regulation of PPARβ observed in response to 1 bout of exercise. We have identified a novel mechanism of regulation of PPARβ expression/action in skeletal muscle with potential physiologic application through the action of α-LA, involving the JNK pathway.

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.

Ageing is characterized by sarcopenia partially explained by a decreased sensitivity of protein metabolism to anabolic stimuli such as insulin or amino acids (leucine). Animals fed a n-3PUFA-enriched-diet have shown an increased of whole body protein metabolism sensitivity to insulin in bovine (Gingras et al.2007) associated with an increased of signalling pathways linked to protein metabolism (i.e mTOR). Smith et al, 2011 have confirmed that n-3PUFA also improved mTOR signalling pathways in elderly muscle. However, in this former study, the effect on insulin relatively to amino acid sensitivity remains unknown since the improvement of muscle protein synthesis was shown under a hyper-aminoacidemic-hyperinsulinic clamp. The present work aimed at demonstrating if protein metabolism sensitivity to insulin alone and/or amino acids (leucine) alone was improved in old rat muscle with a chronic n-3PUFA supplementation. 30 rats (16 month-old) were submitted to a control diet or a n-3PUFA enriched diet for 4 months. The response of muscle protein synthesis and proteolysis to insulin (0,1,5,75nM) or to leucine (0,100,200 m M) was measured in vitro on epitrochlearis muscles. Our results showed that protein synthesis stimulation or proteolysis inhibi- tion by leucine was not changed when animals were dietary supplemented

Aims/hypothesis: Inflammation and ectopic lipid deposition contribute to obesity-related insulin resistance (IR). Studies have shown that deficiency of the proinflammatory cytokine tumor necrosis factor-alpha (INF alpha) protects against the IR induced by a high-fat diet (HFD). We aimed to evaluate the relationship between HFD-related inflammation and lipid deposition in skeletal muscle and liver. Experimental design: Wild-type (WT) and TNF alpha-deficient (TNF alpha-KO) mice were subjected to an HFD for 12 weeks. A glucose tolerance test was performed to evaluate IR. Inflammatory status was assessed by measuring plasma and tissue transcript levels of cytokines. Lipid intermediate concentrations were measured in plasma, muscle and liver. The expression of genes involved in fatty acid transport, synthesis and oxidation was analyzed in adipose tissue, muscle and liver. Results: HFD induced a higher body weight gain in TNF alpha-KO mice than in WT mice. The weight of epididymal and abdominal adipose tissues was twofold lower in WT mice than in TNF alpha-KO mice, whereas liver weight was significantly heavier in WT mice. IR, systemic and adipose tissue inflammation, and plasma nonesterified fatty acid levels were reduced in TNF alpha-KO mice fed an HFD. INFa deficiency improved fatty acid metabolism and had a protective effect against lipid deposition, inflammation and fibrosis associated with HFD in liver but had no impact on these markers in muscle. Conclusions: Our data suggest that in an HFD context, TNF alpha deficiency reduced hepatic lipid accumulation through two mechanisms: an increase in adipose tissue storage capacity and a decrease in fatty acid uptake and synthesis in the liver. (C) 2012 Elsevier Inc. All rights reserved.

Leucine acts as a signal nutrient in promoting protein synthesis in skeletal muscle and adipose tissue via mTOR pathway activation, and may be of interest in age-related sarcopenia. However, hyper-activation of mTOR/S6K1 has been suggested to inhibit the first steps of insulin signaling and finally promote insulin resistance. The impact of long-term dietary leucine supplementation on insulin signaling and sensitivity was investigated in old rats (18 months old) fed a 15% protein diet supplemented (LEU group) or not (C group) with 4.5% leucine for 6 months. The resulting effects on muscle and fat were examined. mTOR/S6K1 signaling pathway was not significantly altered in muscle from old rats subjected to long-term dietary leucine excess, whereas it was increased in adipose tissue. Overall glucose tolerance was not changed but insulin-stimulated glucose transport was improved in muscles from leucine-supplemented rats related to improvement in Akt expression and phosphorylation in response to food intake. No change in skeletal muscle mass was observed, whereas perirenal adipose tissue mass accumulated (+45%) in leucine-supplemented rats. A prolonged leucine supplementation in old rats differently modulates mTOR/S6K pathways in muscle and adipose tissue. It does not increase muscle mass but seems to promote hypertrophy and hyperplasia of adipose tissue that did not result in insulin resistance.

International audience; Background & aims: Age-related inflammation and insulin resistance (IR) have been implicated in the inability of old muscles to properly respond to anabolic stimuli such as amino acids (AA) or insulin. Since fatty acids can modulate inflammation and IR in muscle cells, we investigated the effect of palmitate-enriched diet and oleate-enriched diet on inflammation, IR and muscle protein synthesis (MPS) rate in old rats.Methods: Twenty-four 25-month-old rats were fed either a control diet (OC), an oleate-enriched diet (HFO) or a palmitate-enriched diet (HFP) for 16 weeks. MPS using labeled amino acids and mTOR activation were assessed after AA and insulin anabolic stimulation to mimic postprandial state.Results: IR and systemic and adipose tissue inflammation (TNF alpha. and IL1 beta) were improved in the HFO group. Muscle genes controlling mitochondrial beta-oxidation (PPARs, MCAD and CPT-1b) were up-regulated in the HFO group. AA and insulin-stimulated MPS in the HFO group only, and this stimulation was related to activation of the Akt/mTOR pathway.Conclusions: The age-related MPS response to anabolic signals was improved in rats fed an oleate-enriched diet. This effect was related to activation of muscle oxidative pathways, lower IR, and a decrease in inflammation.

Although activation of the mammalian target of rapamycin complex/p70 S6 kinase (S6K1) pathway by leucine is efficient to stimulate muscle protein synthesis, it can also exert inhibition on the early steps of insulin signaling leading to insulin resistance. We investigated the impact of 5-week leucine supplementation on insulin signaling and sensitivity in 4-month old rats fed a 15% protein diet supplemented (LEU) or not (C) with 4.5% leucine. An oral glucose tolerance test was performed in each rat at the end of the supplementation and glucose transport was measured in vitro using isolated epitrochlearis muscles incubated with 2-deoxy-D-[(3)H]-glucose under increasing insulin concentrations. Insulin signaling was assessed on gastrocnemius at the postabsorptive state or 30 and 60 min after gavage with a nutrient bolus. Tyrosine phosphorylation of IR beta, IRS1 and PI3 kinase activity were reduced in LEU group 30 min after feeding (-36%, -36% and -38% respectively, P

Beside insulinoma, alternative causes of hyperinsulinaemic hypoglycaemia include the rare autoimmune syndrome related to spontaneous autoantibodies either to insulin or to insulin receptor. We describe a case of hypoglycaemia with high insulinemia in which insulinoma could not be evidenced. Surprisingly, we found in the patient's serum both insulin autoantibodies and insulin receptor autoantibodies. Available data eventually supported the predominant role of insulin autoantibodies rather than insulin receptor autoantibodies in the mechanism of hypoglycaemia of this patient. Insulin antibodies were present in high titre. Most of the insulin in serum was bound to the insulin antibodies and free insulin was slightly increased. HLA typing displayed DR4 haplotype, known to be strongly linked to the insulin autoimmune syndrome. The patient's serum was able to inhibit insulin binding to its receptor in a cultured cell line overexpressing insulin receptors both in experiments with native serum and with serum depleted from insulin antibodies. However, we could not demonstrate that the insulin receptor antibodies had insulin mimicking effect. We have no obvious explanation for the presence of these two antibodies in the same patient. Possible hypotheses might involve an idiotype-anti-idiotype mechanism or a poly-autoimmune disease.

The phosphorylation of a previously uncharacterized protein of apparent M r ≈ 140,000 was found to be increased when rat adipocytes were incubated with insulin. The sequences of peptides generated by digesting the protein with trypsin matched perfectly with sequences in mouse lipin. Lipin is the product of the gene that is mutated in fatty liver dystrophy ( fld ) mice [Peterfy, M., Phan, J., Xu, P. & Reue, K (2001) Nat. Genet. 27, 121–124], which exhibit several phenotypic abnormalities including hyperlipidemia, defects in adipocyte differentiation, impaired glucose tolerance, and slow growth. When immunoblots were prepared with lipin antibodies, both endogenous adipocyte lipin and recombinant lipin overexpressed in HEK293 cells appeared as bands ranging in apparent M r from 120,000 to 140,000. Incubating adipocytes with insulin decreased the electrophoretic mobility and stimulated the phosphorylation of both Ser and Thr residues in lipin. The effects of insulin were abolished by inhibitors of phosphatidylinositol 3-OH kinase, and by rapamycin, a specific inhibitor of the mammalian target of rapamcyin (mTOR). The inhibition by rapamycin was blocked by FK506, which competitively inhibits those effects of rapamycin that are mediated by inhibition of mTOR. Moreover, amino acids, which activate mTOR, mimicked insulin by increasing lipin phosphorylation in a rapamycin-sensitive manner. Thus, lipin represents a target of the mTOR pathway, and potentially links this nutrient-sensing pathway to adipocyte development.

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Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.