Dietary fatty acid composition drives neuroinflammation and impaired behavior in obesity.

Nutrient composition in obesogenic diets may influence the severity of disorders associated with obesity such as insulin-resistance and chronic inflammation. Here we hypothesized that obesogenic diets rich in fat and varying in fatty acid composition, particularly in omega 6 (ω6) to omega 3 (ω3) ratio, have various effects on energy metabolism, neuroinflammation and behavior. Mice were fed either a control diet or a high fat diet (HFD) containing either low (LO), medium (ME) or high (HI) ω6/ω3 ratio. Mice from the HFD-LO group consumed less calories and exhibited less body weight gain compared to other HFD groups. Both HFD-ME and HFD-HI impaired glucose metabolism while HFD-LO partly prevented insulin intolerance and was associated with normal leptin levels despite higher subcutaneous and perigonadal adiposity. Only HFD-HI increased anxiety and impaired spatial memory, together with increased inflammation in the hypothalamus and hippocampus. Our results show that impaired glucose metabolism and neuroinflammation are uncoupled, and support that diets with a high ω6/ω3 ratio are associated with neuroinflammation and the behavioral deterioration coupled with the consumption of diets rich in fat.

The Association between Oxytocin and Lower Limb Osteoarthritis: A Prospective Cohort Study.

Oxytocin (OT), a neuropeptide best known for its role in emotional and social behaviors, has been linked to osteoarthritis (OA). This study aimed to investigate the serum OT level in hip and/or knee OA patients and to study its association with disease progression. Patients from the KHOALA cohort with symptomatic hip and/or knee OA (Kellgren and Lawrence (KL) scores of 2 and 3) and follow-up at 5 years were included in this analysis. The primary endpoint was structural radiological progression, which was defined as an increase of at least one KL point at 5 years. Logistic regression models were used to estimate the associations between OT levels and KL progression while controlling for gender, age, BMI, diabetes and leptin levels. Data from 174 hip OA patients and 332 knee OA patients were analyzed independently. No differences in OT levels were found between the 'progressors' and 'non-progressors' groups among the hip OA patients and knee OA patients, respectively. No statistically significant associations were found between the OT levels at baseline and KL progression at 5 years, the KL score at baseline or the clinical outcomes. Higher structural damage at baseline and severe structural progression of hip and knee osteoarthritis did not appear to be associated with a low serum OT level at baseline.

FRIZZY PANICLE drives supernumerary spikelets in bread wheat.

Bread wheat (Triticum aestivum) inflorescences, or spikes, are characteristically unbranched and normally bear one spikelet per rachis node. Wheat mutants on which supernumerary spikelets (SSs) develop are particularly useful resources for work towards understanding the genetic mechanisms underlying wheat inflorescence architecture and, ultimately, yield components. Here, we report the characterization of genetically unrelated mutants leading to the identification of the wheat FRIZZY PANICLE (FZP) gene, encoding a member of the APETALA2/Ethylene Response Factor transcription factor family, which drives the SS trait in bread wheat. Structural and functional characterization of the three wheat FZP homoeologous genes (WFZP) revealed that coding mutations of WFZP-D cause the SS phenotype, with the most severe effect when WFZP-D lesions are combined with a frameshift mutation in WFZP-A. We provide WFZP-based resources that may be useful for genetic manipulations with the aim of improving bread wheat yield by increasing grain number.

Biphasic Calcium Phosphate and Activated Carbon Microparticles in a Plasma Clot for Bone Reconstruction and In Situ Drug Delivery: A Feasibility Study.

The development of bone-filling biomaterials capable of delivering in situ bone growth promoters or therapeutic agents is a key area of research. We previously developed a biomaterial constituting biphasic calcium phosphate (BCP) microparticles embedded in an autologous blood or plasma clot, which induced bone-like tissue formation in ectopic sites and mature bone formation in orthotopic sites, in small and large animals. More recently, we showed that activated carbon (AC) fiber cloth is a biocompatible material that can be used, due to its multiscale porosity, as therapeutic drug delivery system. The present work aimed first to assess the feasibility of preparing calibrated AC microparticles, and second to investigate the properties of a BCP/AC microparticle combination embedded in a plasma clot. We show here, for the first time, after subcutaneous (SC) implantation in mice, that the addition of AC microparticles to a BCP/plasma clot does not impair bone-like tissue formation and has a beneficial effect on the vascularization of the newly formed tissue. Our results also confirm, in this SC model, the ability of AC in particle form to adsorb and deliver large molecules at an implantation site. Altogether, these results demonstrate the feasibility of using this BCP/AC/plasma clot composite for bone reconstruction and drug delivery.

Long-Term Fate and Efficacy of a Biomimetic (Sr)-Apatite-Coated Carbon Patch Used for Bone Reconstruction.

Critical bone defect repair remains a major medical challenge. Developing biocompatible materials with bone-healing ability is a key field of research, and calcium-deficient apatites (CDA) are appealing bioactive candidates. We previously described a method to cover activated carbon cloths (ACC) with CDA or strontium-doped CDA coatings to generate bone patches. Our previous study in rats revealed that apposition of ACC or ACC/CDA patches on cortical bone defects accelerated bone repair in the short term. This study aimed to analyze in the medium term the reconstruction of cortical bone in the presence of ACC/CDA or ACC/10Sr-CDA patches corresponding to 6 at.% of strontium substitution. It also aimed to examine the behavior of these cloths in the medium and long term, in situ and at distance. Our results at day 26 confirm the particular efficacy of strontium-doped patches on bone reconstruction, leading to new thick bone with high bone quality as quantified by Raman microspectroscopy. At 6 months the biocompatibility and complete osteointegration of these carbon cloths and the absence of micrometric carbon debris, either out of the implantation site or within peripheral organs, was confirmed. These results demonstrate that these composite carbon patches are promising biomaterials to accelerate bone reconstruction.

Effects of Fatty Acid Metabolites on Adipocytes Britening: Role of Thromboxane A2.

Obesity is a complex disease highly related to diet and lifestyle and is associated with low amount of thermogenic adipocytes. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to fight overweight and associated comorbidities. Recent studies suggest a role for several fatty acids and their metabolites, called lipokines, in the control of thermogenesis. The purpose of this work was to analyze the role of several lipokines in the control of brown/brite adipocyte formation. We used a validated human adipocyte model, human multipotent adipose-derived stem cell model (hMADS). In the absence of rosiglitazone, hMADS cells differentiate into white adipocytes, but convert into brite adipocytes upon rosiglitazone or prostacyclin 2 (PGI2) treatment. Gene expression was quantified using RT-qPCR and protein levels were assessed by Western blotting. We show here that lipokines such as 12,13-diHOME, 12-HEPE, 15dPGJ2 and 15dPGJ3 were not able to induce browning of white hMADS adipocytes. However, both fatty acid esters of hydroxy fatty acids (FAHFAs), 9-PAHPA and 9-PAHSA potentiated brown key marker UCP1 mRNA levels. Interestingly, CTA2, the stable analog of thromboxane A2 (TXA2), but not its inactive metabolite TXB2, inhibited the rosiglitazone and PGI2-induced browning of hMADS adipocytes. These results pinpoint TXA2 as a lipokine inhibiting brown adipocyte formation that is antagonized by PGI2. Our data open new horizons in the development of potential therapies based on the control of thromboxane A2/prostacyclin balance to combat obesity and associated metabolic disorders.

Cold-stimulated brown adipose tissue activation is related to changes in serum metabolites relevant to NAD+ metabolism in humans.

Cold-induced brown adipose tissue (BAT) activation is considered to improve metabolic health. In murine BAT, cold increases the fundamental molecule for mitochondrial function, nicotinamide adenine dinucleotide (NAD+), but limited knowledge of NAD+ metabolism during cold in human BAT metabolism exists. We show that cold increases the serum metabolites of the NAD+ salvage pathway (nicotinamide and 1-methylnicotinamide) in humans. Additionally, individuals with cold-stimulated BAT activation have decreased levels of metabolites from the de novo NAD+ biosynthesis pathway (tryptophan, kynurenine). Serum nicotinamide correlates positively with cold-stimulated BAT activation, whereas tryptophan and kynurenine correlate negatively. Furthermore, the expression of genes involved in NAD+ biosynthesis in BAT is related to markers of metabolic health. Our data indicate that cold increases serum tryptophan conversion to nicotinamide to be further utilized by BAT. We conclude that NAD+ metabolism is activated upon cold in humans and is probably regulated in a coordinated fashion by several tissues.

The adiponectin receptor agonist AdipoRon normalizes glucose metabolism and prevents obesity but not growth retardation induced by glucocorticoids in young mice.

OBJECTIVE: Glucocorticoids (GCs) are highly effective anti-inflammatory and immunosuppressive drugs. However, prolonged GC therapy may cause numerous adverse effects leading to diabetes and obesity, as well as bone disorders such as osteoporosis in adults and growth retardation in children and adolescents. Prevention and care of the GC-induced adverse effects remain challenging. We have previously demonstrated the efficacy of a treatment with a non-peptidic agonist of adiponectin receptors, AdipoRon, to reverse behaviour disorders and fat mass gain induced by long-term GC treatment. In this work, we have established a relevant model of GC-induced growth and metabolic disorders and determined that AdipoRon is a potential therapeutic tool to reverse these metabolic disturbances. METHODS: 5-Week-old mice were treated continuously with or without corticosterone (35 mg/L) in drinking water for seven consecutive weeks. Taking advantage of this mouse model displaying various growth and metabolic disorders, we assayed whether AdipoRon (daily intraperitoneal injection of 1 mg/kg/day for the last 20 days) might prevent the GC-induced adverse effects. The control group was treated with vehicle only. Nutritional behaviors and metabolic parameters were followed-up throughout the treatment. Serum insulin and leptin levels were measured by ELISA. Computed tomography and histological analysis of adipose tissue were assessed at the end of the experimental procedure. RESULTS: We found that GC treatment in young mice resulted in continuously increased body weight gain associated with a food intake increase. Compared to vehicle-, GC-treated mice displayed early major hyperleptinemia (up to 6-fold more) and hyperinsulinemia (up to 20-fold more) maintained throughout the treatment. At the end of the experimental procedure, GC-treated mice displayed bone growth retardation (e.g. femur length 15.1 versus 14.0 mm, P < 0.01), higher abdominal adipose tissue volume (4.1 versus 2.3, P < 0.01) and altered glucose metabolism compared to control mice. Interestingly, AdipoRon prevented GC-induced effects on energy metabolism such as abdominal adiposity, insulinemia and leptinemia. However, AdipoRon failed to counteract bone growth retardation. CONCLUSION: We characterized the very early pathological steps induced by long-term GC in young mice in a relevant model, including growth retardation, fat mass gain and glucose homeostasis dysregulation. The adiponectin system stimulation enabled normalization of the adipose tissue and metabolic features of GC-treated mice. Adiponectin receptor agonists such as AdipoRon might constitute a novel way to counteract some GC-induced adverse effects.

The Rosmarinus Bioactive Compound Carnosic Acid Is a Novel PPAR Antagonist That Inhibits the Browning of White Adipocytes.

Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect ß-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.

microRNA-375 regulates glucose metabolism-related signaling for insulin secretion.

Enhanced beta cell glycolytic and oxidative metabolism are necessary for glucose-induced insulin secretion. While several microRNAs modulate beta cell homeostasis, miR-375 stands out as it is highly expressed in beta cells where it regulates beta cell function, proliferation and differentiation. As glucose metabolism is central in all aspects of beta cell functioning, we investigated the role of miR-375 in this process using human and rat islets; the latter being an appropriate model for in-depth investigation. We used forced expression and repression of mR-375 in rat and human primary islet cells followed by analysis of insulin secretion and metabolism. Additionally, miR-375 expression and glucose-induced insulin secretion were compared in islets from rats at different developmental ages. We found that overexpressing of miR-375 in rat and human islet cells blunted insulin secretion in response to glucose but not to α-ketoisocaproate or KCl. Further, miR-375 reduced O2 consumption related to glycolysis and pyruvate metabolism, but not in response to α-ketoisocaproate. Concomitantly, lactate production was augmented suggesting that glucose-derived pyruvate is shifted away from mitochondria. Forced miR-375 expression in rat or human islets increased mRNA levels of pyruvate dehydrogenase kinase-4, but decreased those of pyruvate carboxylase and malate dehydrogenase1. Finally, reduced miR-375 expression was associated with maturation of fetal rat beta cells and acquisition of glucose-induced insulin secretion function. Altogether our findings identify miR-375 as an efficacious regulator of beta cell glucose metabolism and of insulin secretion, and could be determinant to functional beta cell developmental maturation.

miR-139 impacts FoxO1 action by decreasing FoxO1 protein in mouse hepatocytes.

FoxO1 is a master regulator of signaling pathways used by growth factors and hormones, including insulin. Its activity is regulated by changes in subcellular localization coupled to post-translational modifications such as phosphorylation, ubiquitination, and acetylation. As microRNAs have emerged as a newly identified means by which cells fine-tune gene expression, we hypothesized that they could regulate FoxO1. Since FoxO1 plays a key role in the liver, we used immortalized neonatal mouse hepatocytes to analyze the effects of potential microRNAs targeting FoxO1. We found that miR-139 targets FoxO1 mRNA directly and reduces the level of the protein without affecting transcript levels. This decrease in FoxO1 protein results in a decrease of its target genes, such as AdQR1, AdQR2 and Mttp. Our findings suggest a novel mode of FoxO1 regulation by which miR-139 could maintain the protein level of FoxO1 to preserve homeostatic regulation of its transcriptional activity in response to environmental stimuli.

Argonaute-2 is associated to brown adipose tissue activation.

MicroRNAs (miRNAs) are important modulators of thermogenic brown adipose tissue (BAT). They have been involved in its differentiation and hence its functioning. While different regulators of the miRNA machinery have been shown to be essential for BAT differentiation, little is known about their implication in BAT activation. The aim of this work was to evaluate the role of AGO2, the chief miRNA mediator, in BAT activation. We took advantage of two non-genetic models of BAT activation to analyze the miRNA machinery and miRNA expression in BAT. We used principal component analysis (PCA) to obtain an overview of miRNA expression according to the BAT activation state. In vitro, we examined AGO2 expression during brown adipocyte differentiation and activation. Finally, we downregulated AGO2 to reveal its potential role in the thermogenic function of brown adipocytes. PCA analysis allowed to cluster animals on their miRNA signature in active BAT. Moreover, hierarchical clustering showed a positive correlation between global upregulation of miRNA expression and active BAT. Consistently, the miRNA machinery, particularly AGO2, was upregulated in vivo in active BAT and in vitro in mature brown adipocytes. Finally, the partial loss-of-function of AGO2 in mature brown adipocytes is sufficient to lead to a diminished expression of UCP1 associated to a decreased uncoupled respiration. Therefore, our study shows the potential contribution of AGO2 in BAT activation. Since BAT is a calorie-burning tissue these data have a translational potential in terms of therapeutic target in the field of altered fuel homeostasis associated to obesity and diabetes.

Amino acid-induced regulation of hepatocyte growth: possible role of Drosha.

In an adult healthy liver, hepatocytes are in a quiescent stage unless a physical injury, such as ablation, or a toxic attack occur. Indeed, to maintain their crucial organismal homeostatic role, the damaged or remaining hepatocytes will start proliferating to restore their functional mass. One of the limiting conditions for cell proliferation is amino-acid availability, necessary both for the synthesis of proteins important for cell growth and division, and for the activation of the mTOR pathway, known for its considerable role in the regulation of cell proliferation. The overarching aim of our present work was to investigate the role of amino acids in the regulation of the switch between quiescence and growth of adult hepatocytes. To do so we used non-confluent primary adult rat hepatocytes as a model of partially ablated liver. We discovered that the absence of amino acids induces in primary rat hepatocytes the entrance in a quiescence state together with an increase in Drosha protein, which does not involve the mTOR pathway. Conversely, Drosha knockdown allows the hepatocytes, quiescent after amino-acid deprivation, to proliferate again. Further, hepatocyte proliferation appears to be independent of miRNAs, the canonical downstream partners of Drosha. Taken together, our observations reveal an intriguing non-canonical action of Drosha in the control of growth regulation of adult hepatocytes responding to a nutritional strain, and they may help to design novel preventive and/or therapeutic approaches for hepatic failure.

Nuclear forkhead box O1 controls and integrates key signaling pathways in hepatocytes.

Insulin inhibits forkhead O class (FoxO) transcription factors, which down-regulate the expression of genes involved in metabolism, cell cycle arrest, and apoptosis. After being phosphorylated by protein kinase B (PKB) on S253 in its DNA-binding domain, Foxo1 is phosphorylated on T24 and additional sites, which overall triggers its nuclear exclusion. During this process, Foxo1 is thought to retain some transcriptional activity and signaling potential. To evaluate this Foxo1 action, we used a Foxo1-ADA mutant that is constitutively nuclear due to mutation of T24 and S316 to A and harbors a mutation of S253 to D. Adenoviral-mediated expression of Foxo1-ADA in hepatocytes activates PKB and MAPK pathways more than expression of wild-type or of a transactivation domain-deleted mutant (Delta256). PKB activation cannot be accounted for by a Foxo1-mediated increase in upstream signaling components such as insulin receptor substrate 1 or 2 or by Foxo1-mediated down-regulation of Tribbles homolog 3. In contrast, Foxo1-ADA increases p38 activity, and p38 is required for effects of Foxo1 on PKB, at least in part. We propose that Foxo1 turns on a feed-forward loop, relayed by p38 and acting to amplify both PKB activation and Foxo1 inhibition. To conclude, key signaling pathways are activated in hepatocytes through nuclear Foxo1.

Age-Dependent Control of Energy Homeostasis by Brown Adipose Tissue in Progeny Subjected to Maternal Diet-Induced Fetal Programming.

Epidemiological and animal studies show that deleterious maternal environments predispose aging offspring to metabolic disorders and type 2 diabetes. Young progenies in a rat model of maternal low-protein (LP) diet are normoglycemic despite collapsed insulin secretion. However, without further worsening of the insulin secretion defect, glucose homeostasis deteriorates in aging LP descendants. Here we report that normoglycemic and insulinopenic 3-month-old LP progeny shows increased body temperature and energy dissipation in association with enhanced brown adipose tissue (BAT) activity. In addition, it is protected against a cold challenge and high-fat diet (HFD)-induced obesity with associated insulin resistance and hyperglycemia. Surgical BAT ablation in 3-month-old LP offspring normalizes body temperature and causes postprandial hyperglycemia. At 10 months, BAT activity declines in LP progeny with the appearance of reduced protection to HFD-induced obesity; at 18 months, LP progeny displays a BAT activity comparable to control offspring and insulin resistance and hyperglycemia occur. Together our findings identify BAT as a decisive physiological determinant of the onset of metabolic dysregulation in offspring predisposed to altered ß-cell function and hyperglycemia and place it as a critical regulator of fetal programming of adult metabolic disease.

Visfatin expression analysis in association with recruitment and activation of human and rodent brown and brite adipocytes.

Human brown adipocytes are able to burn fat and glucose and are now considered as a potential strategy to treat obesity, type 2 diabetes and metabolic disorders. Besides their thermogenic function, brown adipocytes are able to secrete adipokines. One of these is visfatin, a nicotinamide phosphoribosyltransferase involved in nicotinamide dinucleotide synthesis, which is known to participate in the synthesis of insulin by pancreatic ß cells. In a therapeutic context, it is of interest to establish whether a potential correlation exists between brown adipocyte activation and/or brite adipocyte recruitment, and adipokine expression. We analyzed visfatin expression, as a pre-requisite to its secretion, in rodent and human biopsies and cell models of brown/brite adipocytes. We found that visfatin was preferentially expressed in mature adipocytes and that this expression was higher in brown adipose tissue of rodents compared to other fat depots. However, using various rodent models we were unable to find any correlation between visfatin expression and brown or brite adipocyte activation or recruitment. Interestingly, the situation is different in humans where visfatin expression was found to be equivalent between white and brown or brite adipocytes in vivo and in vitro. In conclusion, visfatin can be considered only as a rodent brown adipocyte biomarker, independently of tissue activation.

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.

Maternal protein restriction leads to pancreatic failure in offspring: role of misexpressed microRNA-375.

The intrauterine environment of the fetus is a preeminent actor in long-term health. Indeed, mounting evidence shows that maternal malnutrition increases the risk of type 2 diabetes (T2D) in progeny. Although the consequences of a disturbed prenatal environment on the development of the pancreas are known, the underlying mechanisms are poorly defined. In rats, restriction of protein during gestation alters the development of the endocrine pancreas and favors the occurrence of T2D later in life. Here we evaluate the potential role of perturbed microRNA (miRNA) expression in the decreased ß-cell mass and insulin secretion characterizing progeny of pregnant dams fed a low-protein (LP) diet. miRNA profiling shows increased expression of several miRNAs, including miR-375, in the pancreas of fetuses of mothers fed an LP diet. The expression of miR-375 remains augmented in neoformed islets derived from fetuses and in islets from adult (3-month-old) progeny of mothers fed an LP diet. miR-375 regulates the proliferation and insulin secretion of dissociated islet cells, contributing to the reduced ß-cell mass and function of progeny of mothers fed an LP diet. Remarkably, miR-375 normalization in LP-derived islet cells restores ß-cell proliferation and insulin secretion. Our findings suggest the existence of a developmental memory in islets that registers intrauterine protein restriction. Hence, pancreatic failure after in utero malnutrition could result from transgenerational transmission of miRNA misexpression in ß-cells.

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.