[Thermoneutrality and animal study]. / Thermoneutralité chez la souris et expérimentation animale.

Animal models remain important for the study of human pathologies. The most widely used model (mouse) is an endothermic mammal like humans, maintained at ambient temperatures (22 °C). Its energy metabolism is overactivated, a situation rarely observed in humans thanks to various adaptations (clothing, heating…). The thermoneutral zone is defined as a range of ambient temperatures that allows an organism to regulate body temperature without using additional thermoregulatory processes. There are many examples of divergent results between studies conducted at 22 °C or at 30 °C (thermoneutrality for mice). Therefore, it seems essential to take into account the housing temperature both for animal welfare and for the relevance of the results.

Title: Thermoneutralité chez la souris et expérimentation animale. Abstract: Les modèles animaux demeurent une nécessité pour l'étude des maladies humaines. Le modèle le plus utilisé, la souris, est, comme les êtres humains, un mammifère endotherme maintenu à des températures ambiantes (22 °C). Son métabolisme énergétique est donc suractivé, une situation rarement observée chez les êtres humains grâce à diverses adaptations (vêtements, chauffage, etc.). La zone de thermoneutralité est définie comme une plage de températures ambiantes qui permet à un organisme de réguler sa température corporelle sans recourir à des processus de thermorégulation supplémentaires. Il existe de nombreux exemples de résultats divergents entre des études menées à 22 °C et celles réalisées à 30 °C (thermoneutralité chez la souris). Il semble donc essentiel de prendre en compte la température d'hébergement tant pour le bien-être animal que pour la pertinence des résultats des expériences réalisées.

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples.

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARß/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARß/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARß/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARß/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARß/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARß/δ activation was combined with exercise training. Lastly, PPARß/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARß/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.

Invalidation of the Transcriptional Modulator of Lipid Metabolism PPARß/δ in T Cells Prevents Age-Related Alteration of Body Composition and Loss of Endurance Capacity.

Anti-inflammatory regulatory T cells (Tregs) are the most metabolically flexible CD4+ T cells by using both glycolysis and fatty acid oxidation (FAO) which allow them to migrate in tissues. With aging, Tregs accumulate in secondary lymphoid organs and are involved in impairment of skeletal muscle (SKM) regeneration and mass maintenance. In this study, we showed that a deletion of a FAO modulator, peroxisome proliferator-activated receptor beta/delta (PPARß/δ), specifically in T cells (KO-T PPARß/δ), increased the number of CD4+ T cells at day 2 following a cardiotoxin-induced SKM regeneration. Older KO-T PPARß/δ mice maintained a Tregs prevalence in lymph nodes similar to young mice. Surprisingly, KO-T PPARß/δ mice were protected from the effects of age on lean and fat mass and endurance capacity. Our results lead us to propose an original potential role of T cell metabolism in the effects of aging on the maintenance of body composition and endurance capacity.

Alpha-lipoic acid supplementation increases the efficacy of exercise- and diet-induced obesity treatment and induces immunometabolic changes in female mice and women.

The decrease in the regulatory T cells (Tregs) population is highly involved in adipose tissue inflammation and insulin resistance in obesity. Tregs depend on fatty acids via ß-oxidation for immunosuppressive function adapting their antioxidant systems to allow survival to oxidative stress. In this study, we have hypothesized that a dietary supplementation with alpha-lipoic acid (ALA), a powerful antioxidant, would improve immunometabolism when added to the classical strategy of obesity treatment. First, we showed by in vitro experiments that ALA favors the polarization of mice CD4 + T cells toward Tregs. Next, we have carried out a translational study where female obese mice and women were supplemented with ALA or vehicle/placebo (mice: 2.5 gALA /kgfood ; 6 weeks; women: 600 mgALA /day, 8 weeks) while following a protocol including regular exercise and a change in diet. Fatty acid oxidation potential and activity of nuclear erythroid-related factor 2 (NRF2) of mouse secondary lymphoid tissues were improved by ALA supplementation. ALA reduced visceral adipose tissue (VAT) mass and preserved Tregs in VAT in mice. In women, ALA supplementation induced significant metabolic changes of circulating CD4 + T cells including increased oxidative capacity and fatty acid oxidation, ameliorated their redox status, and improved the reduction of visceral fat mass. While appropriate biological markers are still required to be used in clinics to judge the effectiveness of long-term obesity treatment, further studies in female mice and women are needed to determine whether these immunometabolic changes would reduce VAT mass-associated risk for secondary health issues arising from obesity.

Complementary Immunometabolic Effects of Exercise and PPARß/δ Agonist in the Context of Diet-Induced Weight Loss in Obese Female Mice.

Regular aerobic exercise, independently of weight loss, improves metabolic and anti-inflammatory states, and can be regarded as beneficial in counteracting obesity-induced low-grade inflammation. However, it is still unknown how exercise alters immunometabolism in a context of dietary changes. Agonists of the Peroxisome Proliferator Activated-Receptor beta/delta (PPARß/δ) have been studied this last decade as "exercise-mimetics", which are potential therapies for metabolic diseases. In this study, we address the question of whether PPARß/δ agonist treatment would improve the immunometabolic changes induced by exercise in diet-induced obese female mice, having switched from a high fat diet to a normal diet. 24 mice were assigned to groups according to an 8-week exercise training program and/or an 8-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist. Our results show metabolic changes of peripheral lymphoid tissues with PPARß/δ agonist (increase in fatty acid oxidation gene expression) or exercise (increase in AMPK activity) and a potentiating effect of the combination of both on the percentage of anti-inflammatory Foxp3+ T cells. Those effects are associated with a decreased visceral adipose tissue mass and skeletal muscle inflammation (TNF-α, Il-6, Il-1ß mRNA level), an increase in skeletal muscle oxidative capacities (citrate synthase activity, endurance capacity), and insulin sensitivity. We conclude that a therapeutic approach targeting the PPARß/δ pathway would improve obesity treatment.

Decrease in αß/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

The implication of αß 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. αß 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 ß (PPAR-ß) specifically in T cells [transgenic (Tg) T-PPAR-ß] that exhibits a partial depletion in αß T cells and no change in γδ T-cell number. This results in a decreased αß/γδ T-cell ratio in lymphoid organs. We now show that Tg T-PPAR-ß 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 αß/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 αß/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

Peroxisome Proliferator Activated Receptor Beta (PPARß) activity increases the immune response and shortens the early phases of skeletal muscle regeneration.

Peroxisome Proliferator-Activated Receptor Beta (PPARß) is a transcription factor playing an important role in both muscle myogenesis and remodeling, and in inflammation. However, its role in the coordination of the transient muscle inflammation and reparation process following muscle injury has not yet been fully determined. We postulated that activation of the PPARß pathway alters the early phase of the muscle regeneration process, i.e. when immune cells infiltrate in injured muscle. Tibialis anteriors of C57BL6/J mice treated or not with the PPARß agonist GW0742 were injected with cardiotoxin (or with physiological serum for the contralateral muscle). Muscle regeneration was monitored on days 4, 7, and 14 post-injury. We found that treatment of mice with GW0742 increased, at day 4 post-damage, the recruitment of immune cells (M1 and M2 macrophages) and upregulated the expression of the anti-inflammatory cytokine IL-10 and TGF-ß mRNA. Those effects were accompanied by a significant increase at day 4 of myogenic regulatory factors (Pax7, MyoD, Myf5, Myogenin) mRNA in GW0742-treated mice. However, we showed an earlier return (7 days vs 14 days) of Myf5 and Myogenin to basal levels in GW0742- compared to DMSO-treated mice. Differential effects of GW0742 observed during the regeneration were associated with variations of PPARß pathway activity. Collectively, our findings indicate that PPARß pathway activity shortens the early phases of skeletal muscle regeneration by increasing the immune response.

A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development.

Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor ß (PPARß) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARß activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4-CD8- double-negative stage 4 (DN4) thymocytes. These results support a model where PPARß activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αß T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells.

α-Lipoic acid up-regulates expression of peroxisome proliferator-activated receptor ß in skeletal muscle: involvement of the JNK signaling pathway.

We hypothesized that α-lipoic acid (α-LA) might interact with the transcriptional control of peroxisome proliferator-activated receptor (PPAR)ß in skeletal muscle. Molecular mechanisms were investigated using differentiated C2C12 myotubes treated with α-LA and/or PPARß 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 PPARß but regulates PPARß target gene expression through an active PPARß 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 PPARß mRNA and protein levels (2-fold) to the same extent as with the JNK inhibitor SP600125. Similarly to VWR training effect, PPARß 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 PPARß observed in response to 1 bout of exercise. We have identified a novel mechanism of regulation of PPARß expression/action in skeletal muscle with potential physiologic application through the action of α-LA, involving the JNK pathway.

Adipocytes secrete leukotrienes: contribution to obesity-associated inflammation and insulin resistance in mice.

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.

TNFα gene knockout differentially affects lipid deposition in liver and skeletal muscle of high-fat-diet mice.

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-α (TNFα) 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 TNFα-deficient (TNFα-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 TNFα-KO mice than in WT mice. The weight of epididymal and abdominal adipose tissues was twofold lower in WT mice than in TNFα-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 TNFα-KO mice fed an HFD. TNFα 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, TNFα 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.

Differential effect of long-term leucine supplementation on skeletal muscle and adipose tissue in old rats: an insulin signaling pathway approach.

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.

Oleate-enriched diet improves insulin sensitivity and restores muscle protein synthesis in old rats.

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 (TNFα and IL1ß) were improved in the HFO group. Muscle genes controlling mitochondrial ß-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.

Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling pathway associated with overall impaired glucose tolerance.

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 IRß, IRS1 and PI3 kinase activity were reduced in LEU group 30 min after feeding (-36%, -36% and -38% respectively, P<.05) whereas S6K1, S6rp and 4EBP1 phosphorylations were similar. Overall glucose tolerance was reduced in leucine-supplemented rats and was associated with accumulation of perirenal adipose tissue (+27%, P<.05). Conversely, in vitro insulin-response of muscle glucose transport tended to be improved in leucine-supplemented rats. In conclusion, dietary leucine supplementation in adult rats induced a delay in the postprandial stimulation in the early steps of muscle insulin signaling without muscle resistance on insulin-induced glucose uptake. However, it resulted in overall glucose intolerance linked to increased local adiposity. Further investigations are necessary to clearly define the beneficial and/or deleterious effects of chronic dietary leucine supplementation in healthy subjects.

Effects of age, malnutrition and refeeding on the expression and secretion of ghrelin.

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.

Role of amino acids in insulin signaling in adipocytes and their potential to decrease insulin resistance of adipose tissue.

Recently, our knowledge concerning the role of amino acids in signal transduction in mammals has greatly improved. This significant advance is mainly due to the remarkable discovery that the mammalian target of rapamycin (mTOR) protein kinase, known to be activated in response to a large number of hormones, growth factors and cytokines, is also under the tight control of branched-chain amino acids. Actually, both inputs are necessary to fully activate the mTOR pathway, the main function of which is to increase cell size, via the regulation of translational processes. However, amino acids are able to modulate other biological effects and appear to have unexpected actions, as evidenced by our recent work in rat adipocytes. The aim of this review is to summarize novel findings on the role of mTOR and amino acids in insulin signaling in adipocytes. A possible beneficial impact of the use of amino acids in the treatment of insulin resistance is discussed, and hypotheses about the molecular mechanisms underlying their effect are proposed.

Amino acids and leucine allow insulin activation of the PKB/mTOR pathway in normal adipocytes treated with wortmannin and in adipocytes from db/db mice.

Amino acids are nutrients responsible for mammalian target of rapamycin (mTOR) regulation in mammalian cells. The mTOR protein is mainly known for its role in regulating cell growth, notably via protein synthesis. In addition to amino acids, mTOR is regulated by insulin via a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. mTOR mediates crosstalk between amino acids and insulin signaling. We show that in freshly isolated rat adipocytes, insulin stimulates the phosphorylation of mTOR on serine 2448, a protein kinase B (PKB) consensus phosphorylation site. This site is also phosphorylated by amino acids, which in contrast to insulin do not activate PKB. Moreover, insulin and amino acids have an additive effect on mTOR phosphorylation, indicating that they act via two independent pathways. Importantly, amino acids, notably leucine, permit insulin to stimulate PKB when PI 3-kinase is inhibited. They also rescue glucose transport and the mTOR pathway. Further, leucine alone can improve insulin activation of PKB in db/db mice. Our results define the importance of amino acids in insulin signaling and reveal leucine as a key amino acid in disease situations associated with insulin-resistance in adipocytes.

In rat hepatocytes glucagon increases mammalian target of rapamycin phosphorylation on serine 2448 but antagonizes the phosphorylation of its downstream targets induced by insulin and amino acids.

The major function of mammalian target of rapamycin (mTOR) is the control of cell growth. Insulin and amino acids regulate the mTOR pathway, and both are needed to promote its maximal activation. To further understand mTOR regulation by insulin and amino acids, we have studied the enzyme in primary cultures of hepatocytes. We show that insulin increases mTOR phosphorylation on Ser2448, a consensus phosphorylation site for protein kinase B (PKB). Ser2448 phosphorylation is also increased by amino acids, although they do not activate PKB. Furthermore, insulin and amino acids have an additive effect, indicating that they act through distinct pathways. We also show that phosphorylation of Ser2448 does not seem to modulate in vitro phosphorylation of eukaryotic initiation factor 4E-binding protein 1 by mTOR. However, stimulation of hepatocytes with insulin and amino acids leads to an increase in mTOR kinase activity. Rapamycin has no effect on insulin-, glucagon-, and 8-(4-chlorophenylthio)adenosine-cAMP-induced amino acid transport. Surprisingly, glucagon and 8-(4-chlorophenylthio)adenosine-cAMP, which do not activate PKB, stimulate the phosphorylation on Ser2448 of mTOR. However, glucagon inhibits amino acid- and insulin-induced activation of ribosomal S6 protein kinase 1 and phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1. Our results demonstrate that glucagon, which is not able to activate but rather inhibits the mTOR pathways, stimulates the phosphorylation of mTOR on Ser2448. This finding suggests that phosphorylation of this site might not be sufficient for mTOR kinase activity but is likely to be involved in other functions.

Ser-64 and Ser-111 in PHAS-I are dispensable for insulin-stimulated dissociation from eIF4E.

Insulin stimulates phosphorylation of multiple sites in the eIF4E-binding protein, PHAS-I, leading to dissociation of the PHAS-I.eIF4E complex and to an increase in cap-dependent translation. The Ser-64 and Ser-111 sites have been proposed to have key roles in controlling the association of PHAS-I and eIF4E. To determine whether the effects of insulin require these sites, we assessed the control of PHAS-I proteins having Ala-64 or Ala-111 mutations. The results indicate that phosphorylation of neither site is required for insulin to promote release of PHAS-I from eIF4E. Also, the mutation of Ser-111, which has been proposed to serve as a necessary priming site for the phosphorylation of other sites in PHAS-I, did not affect the phosphorylation of Thr-36/45, Ser-64, or Thr-69. Insulin promoted the release of eIF4E from PHAS-II, a PHAS isoform that lacks the Ser-111 site, but it was without effect on the amount of eIF4E bound to the third isoform, PHAS-III. The results demonstrate that contrary to widely accepted models, Ser-64 and Ser-111 are not required for the control of PHAS-I binding to eIF4E in cells, implicating phosphorylation of the Thr sites in dissociation of the PHAS-I.eIF4E complex. The findings also indicate that PHAS-II, but not PHAS-III, contributes to the control of protein synthesis by insulin.

Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.

The phosphorylation of a previously uncharacterized protein of apparent M(r) approximately 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.