Many cytokines signal through different cell-surface receptors to activate the transcription factor NF-kappaB. Members of the TRAF protein family have been implicated in the activation of NF-kappaB by the tumour-necrosis factor (TNF)-receptor superfamily. Here we report the identification of a new TRAF family member, designated TRAF6. When overexpressed in human 293 cells, TRAF6 activates NF-kappaB. A dominant-negative mutant of TRAF6 inhibits NF-kappaB activation signalled by interleukin-1 (IL-1) but not by TNF. IL-1 treatment of 293 cells induces the association of TRAF6 with IRAK, a serine/threonine kinase that is rapidly recruited to the IL-1 receptor after IL-1 induction. These findings indicate that TRAF proteins may function as signal transducers for distinct receptor families and that TRAF6 participates in IL-1 signalling.

Fatty acid synthesis in the central nervous system is implicated in the control of food intake and energy expenditure. An intermediate in this pathway, malonyl-CoA, mediates these effects. Malonyl-CoA is an established inhibitor of carnitine palmitoyltransferase-1 (CPT1), an outer mitochondrial membrane enzyme that controls entry of fatty acids into mitochondria and, thereby, fatty acid oxidation. CPT1c, a brain-specific enzyme with high sequence similarity to CPT1a (liver) and CPT1b (muscle) was recently discovered. All three CPTs bind malonyl-CoA, and CPT1a and CPT1b catalyze acyl transfer from various fatty acyl-CoAs to carnitine, whereas CPT1c does not. These findings suggest that CPT1c has a unique function or activation mechanism. We produced a targeted mouse knockout (KO) of CPT1c to investigate its role in energy homeostasis. CPT1c KO mice have lower body weight and food intake, which is consistent with a role as an energy-sensing malonyl-CoA target. Paradoxically, CPT1c KO mice fed a high-fat diet are more susceptible to obesity, suggesting that CPT1c is protective against the effects of fat feeding. CPT1c KO mice also exhibit decreased rates of fatty acid oxidation, which may contribute to their increased susceptibility to diet-induced obesity. These findings indicate that CPT1c is necessary for the regulation of energy homeostasis.acetyl-CoA carboxylase ͉ fatty acid synthase ͉ food intake ͉ malonyl-CoA ͉ obesity B ody weight is maintained by regulating food intake and energy expenditure. This balance is monitored by the central nervous system (CNS) in response to cytokine and endocrine signals, including leptin, ghrelin, obestatin, insulin, cholecystokinin, and peptide YY secreted by peripheral tissues. Concomitantly, parallel pathways in the CNS regulate energy balance by monitoring the availability of neuronal energy-rich metabolic substrates. Integration of these signals occurs in the hypothalamus and, ultimately, in higher brain centers where feeding behavior and energy expenditure are adjusted. Two primary indicators of energy surplus, glucose and fatty acids, are also monitored by subsets of hypothalamic neurons that modulate feeding behavior and energy expenditure (1). Fatty acids (2) and de novo fatty acid synthesis from glucose (3) are known to mediate these effects. Indeed, food intake and body weight have been shown to be altered by manipulating the activities of the enzymes involved in fatty acid synthesis, e.g., fatty acid synthase (FAS) (3), malonyl-CoA decarboxylase (4, 5), acetyl-CoA carboxylase (ACC) (6, 7), stearoyl-CoA desaturase (8, 9), and 5Ј-AMP kinase (10, 11).Inhibition of FAS in the CNS, for example, reduces body weight by rapidly provoking a reduction in food intake and an increase in peripheral energy expenditure (3,12). This inhibition can reverse the weight gain caused by diet-induced obesity (13,14) or mutations in leptin (ob͞ob) or its receptor (db͞db) (3, 15), suggesting that it acts independently of STAT3, which is known to be essential for leptin 's action (16, 17). I...

Background and purpose:The lipid phosphatase known as SH2 domain-containing inositol 5′-phosphatase 2 (SHIP2) plays an important role in the regulation of the intracellular insulin signalling pathway. Recent studies have suggested that inhibition of SHIP2 could produce significant benefits in treatment of type 2 diabetes. However, there were no small molecule SHIP2 inhibitors and we, therefore, aimed to identify this type of compound. Experimental approach: The phosphatase assay with malachite green was used for high-throughput screening. The pharmacological profiles of suitable compounds were further characterized in phosphatase assays, cellular assays and oral administration in normal and diabetic (db/db) mice. Key results: During high-throughput screening, AS1949490 was identified as a potent SHIP2 inhibitor (IC50 = 0.62 mM for SHIP2). This compound was also selective for SHIP2 relative to other intracellular phosphatases. In L6 myotubes, AS1949490 increased the phosphorylation of Akt, glucose consumption and glucose uptake. In FAO hepatocytes, AS1949490 suppressed gluconeogenesis. Acute administration of AS1949490 inhibited the expression of gluconeogenic genes in the livers of normal mice. Chronic treatment of diabetic db/db mice with AS1949490 significantly lowered the plasma glucose level and improved glucose intolerance. These in vivo effects were based in part on the activation of intracellular insulin signalling pathways in the liver. Conclusions and implications:This is the first report of a small molecule inhibitor of SHIP2. This compound will help to elucidate the physiological functions of SHIP2 and its involvement in various diseases, such as type 2 diabetes.

Cytokines and endocrine hormones control food intake and energy expenditure and are critical for maintaining homeostasis and averting serious health problems such as those associated with obesity. In addition, a recently identified system for maintaining energy balance utilizes an ancient nutrient-sensing pathway with which the CNS monitors energy needs by assessing current energy surplus/deficit and responding by modulating appetite and peripheral energy expenditure (Wolfgang and Lane 2006b AbstractWhile the brain does not utilize fatty acids as a primary energy source, recent evidence shows that intermediates of fatty acid metabolism serve as hypothalamic sensors of energy status. Increased hypothalamic malonyl-CoA, an intermediate in fatty acid synthesis, is indicative of energy surplus and leads to the suppression of food intake and increased energy expenditure. Malonyl-CoA functions as an inhibitor of carnitine palmitoyltransferase 1 (CPT1), a mitochondrial outer membrane enzyme that initiates translocation of fatty acids into mitochondria for oxidation. The mammalian brain expresses a unique homologous CPT1, CPT1c, that binds malonyl-CoA tightly but does not support fatty acid oxidation in vivo, in hypothalamic explants or in heterologous cell culture systems. CPT1c knockout (KO) mice under fasted or refed conditions do not exhibit an altered CNS transcriptome of genes known to be involved in fatty acid metabolism. CPT1c KO mice exhibit normal levels of metabolites and of hypothalamic malonylCoA and fatty acyl-CoA levels either in the fasted or refed states. However, CPT1c KO mice exhibit decreased food intake and lower body weight than wild-type littermates. In contrast, CPT1c KO mice gain excessive body weight and body fat when fed a high-fat diet while maintaining lower or equivalent food intake. Heterozygous mice display an intermediate phenotype. These findings provide further evidence that CPT1c plays a role in maintaining energy homeostasis, but not through altered fatty acid oxidation.

Activation of the transcription factor NF-B by extracellular signals involves its release from the inhibitor protein IB␣ in the cytoplasm and subsequent nuclear translocation. NF-B can also be activated by the anticancer agent camptothecin (CPT), which inhibits DNA topoisomerase (Topo) I activity and causes DNA doublestrand breaks during DNA replication to induce S phasedependent cytotoxicity. Here we show that CPT activates NF-B by a mechanism that is dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. NF-B activation by CPT is dramatically diminished in cytoplasts and in CEM/C2 cells expressing a mutant Topo I protein that fails to bind CPT. This response is intensified in S phase cell populations and is prevented by the DNA polymerase inhibitor aphidicolin. In addition, CPT activation of NF-B involves degradation of cytoplasmic IB␣ by the ubiquitin-proteasome pathway in a manner that depends on the IB kinase complex. Finally, inhibition of NF-B activation augments CPT-induced apoptosis. These findings elucidate the progression of signaling events that initiates in the nucleus with CPT-Topo I interaction and continues in the cytoplasm resulting in degradation of IB␣ and nuclear translocation of NF-B to attenuate the apoptotic response.The NF-B/Rel family of transcription factors regulates expression of genes critical for multiple biological processes, including immune responses, inflammatory reactions, and apoptosis (1-3). In mammalian cells, NF-B exists as dimeric complexes composed of p50, p65 (RelA), c-Rel, RelB, or p52. These proteins share a conserved Rel homology domain that encodes dimerization, DNA binding, and nuclear localization functions. NF-B associates with members of the IB family of proteins, most notably IB␣, which masks the nuclear localization sequence of NF-B and retains it in the cytoplasm (4, 5). Dissociation from IB␣ is essential for NF-B to enter the nucleus and to activate gene expression. Several signaling cascades that control NF-B activation converge at an IB kinase (IKK) 1 complex, responsible for site-specific phosphorylation of IB␣ at serines 32 and 36 (6 -10). Phosphorylation of IB␣ induces multiubiquitination of IB␣ and its subsequent degradation by the ubiquitin-dependent 26 S proteasome (11,12). This sequence of events can be induced without de novo protein synthesis by multiple extracellular stimuli, including tumor necrosis factor ␣ (TNF␣), interleukin-1, phorbol ester (PMA), bacterial lipopolysaccharide (LPS), and others. However, NF-B activation can also be achieved through mechanisms that are distinct from the above IKK-dependent model. These include phosphorylation-independent yet proteasome-mediated IB␣ degradation induced by ultraviolet irradiation (13,14), calpain-dependent degradation of IB␣ by silica and TNF␣ (15, 16), and tyrosine phosphorylation-induced dissociation of IB␣ from NF-B following hypoxia and reoxygenation (17). Thus, depending on the stimuli, NF-B can be activated through multiple distinct regulatory pathways.Activation ...