mTORC1 Is a Major Regulatory Node in the FGF21 Signaling Network in Adipocytes.

FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signaling network responsible for mediating these effects, we quantified dynamic changes in the adipocyte phosphoproteome following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins. A major FGF21-regulated signaling node was mTORC1/S6K. In contrast to insulin, FGF21 activated mTORC1 via MAPK rather than through the canonical PI3K/AKT pathway. Activation of mTORC1/S6K by FGF21 was surprising because this is thought to contribute to deleterious metabolic effects such as obesity and insulin resistance. Rather, mTORC1 mediated many of the beneficial actions of FGF21 in vitro, including UCP1 and FGF21 induction, increased adiponectin secretion, and enhanced glucose uptake without any adverse effects on insulin action. This study provides a global view of FGF21 signaling and suggests that mTORC1 may act to facilitate FGF21-mediated health benefits in vivo.

MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene.

INTRODUCTION: The study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged. METHODS: microRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3' untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets. RESULTS: A large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients. CONCLUSIONS: These studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.

Selective insulin resistance in adipocytes.

Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin.

Direction pathway analysis of large-scale proteomics data reveals novel features of the insulin action pathway.

MOTIVATION: With the advancement of high-throughput techniques, large-scale profiling of biological systems with multiple experimental perturbations is becoming more prevalent. Pathway analysis incorporates prior biological knowledge to analyze genes/proteins in groups in a biological context. However, the hypotheses under investigation are often confined to a 1D space (i.e. up, down, either or mixed regulation). Here, we develop direction pathway analysis (DPA), which can be applied to test hypothesis in a high-dimensional space for identifying pathways that display distinct responses across multiple perturbations. RESULTS: Our DPA approach allows for the identification of pathways that display distinct responses across multiple perturbations. To demonstrate the utility and effectiveness, we evaluated DPA under various simulated scenarios and applied it to study insulin action in adipocytes. A major action of insulin in adipocytes is to regulate the movement of proteins from the interior to the cell surface membrane. Quantitative mass spectrometry-based proteomics was used to study this process on a large-scale. The combined dataset comprises four separate treatments. By applying DPA, we identified that several insulin responsive pathways in the plasma membrane trafficking are only partially dependent on the insulin-regulated kinase Akt. We subsequently validated our findings through targeted analysis of key proteins from these pathways using immunoblotting and live cell microscopy. Our results demonstrate that DPA can be applied to dissect pathway networks testing diverse hypotheses and integrating multiple experimental perturbations. AVAILABILITY AND IMPLEMENTATION: The R package 'directPA' is distributed from CRAN under GNU General Public License (GPL)-3 and can be downloaded from: http://cran.r-project.org/web/packages/directPA/index.html CONTACT: jean.yang@sydney.edu.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.

Cell cycle sensing of oxidative stress in Saccharomyces cerevisiae by oxidation of a specific cysteine residue in the transcription factor Swi6p.

Yeast cells begin to bud and enter the S phase when growth conditions are favorable during the G(1) phase. When subjected to some oxidative stresses, cells delay entry at G(1), allowing repair of cellular damage. Hence, oxidative stress sensing is coordinated with the regulation of cell cycle. We identified a novel function of the cell cycle regulator of Saccharomyces cerevisiae, Swi6p, as a redox sensor through its cysteine residue at position 404. When alanine was substituted at this position, the resultant mutant, C404A, was sensitive to several reactive oxygen species and oxidants including linoleic acid hydroperoxide, the superoxide anion, and diamide. This mutant lost the ability to arrest in G(1) phase upon treatment with lipid hydroperoxide. The Cys-404 residue of Swi6p in wild-type cells was oxidized to a sulfenic acid when cells were subjected to linoleic acid hydroperoxide. Mutation of Cys-404 to Ala abolished the down-regulation of expression of the G(1) cyclin genes CLN1, CLN2, PCL1, and PCL2 that occurred when cells of the wild type were exposed to the lipid hydroperoxide. In conclusion, oxidative stress signaling for cell cycle regulation occurs through oxidation of the G(1)/S-specific transcription factor Swi6p and consequently leads to suppression of the expression of G(1) cyclins and a delay in cells entering the cell cycle.

The critical role of glutathione in maintenance of the mitochondrial genome.

Glutathione (GSH) is a key redox buffer and protectant. Growth (approx. one or two divisions) of cells lacking γ-glutamylcysteine synthetase (gsh1) in the absence of GSH led to irreversible respiratory incompetency in all cells, and after five divisions 75% of cells completely lacked mitochondrial DNA (mtDNA). The level of GSH required to allow continuous growth was distinct from that required to prevent loss of mtDNA. GSH limitation led to a change in the transcript levels of 190 genes, including 30 genes regulated by the Aft1p and/or Aft2p transcription factors, which regulate the cellular response to changes in iron availability. Disruption of AFT1 but not AFT2 in gsh1 cells afforded a protective effect on maintenance of respiratory competency, as did overexpression of GRX3 or GRX4 (encoding monothiol glutaredoxins that act as negative regulators of Aft1p). Importantly, an iron-independent mechanism (~30%) was also observed to mediate GSH-dependent mtDNA loss. Analysis of the redox environment in the cytosol, mitochondrial matrix, and intermembrane space (IMS) found that the cytosol was most severely and rapidly affected by GSH depletion. GSH may also modulate the redox environment of the IMS. The implications of altered GSH homeostasis for maintenance of mtDNA, compartmental redox, and the pathophysiology of certain diseases are discussed.

Akt inhibitors reduce glucose uptake independently of their effects on Akt.

The protein kinase Akt is involved in various cellular processes, including cell proliferation, growth and metabolism. Hyperactivation of Akt is commonly observed in human tumours and so this pathway has been the focus of targeted drug discovery. However, Akt also plays an essential role in other physiological processes, such as the insulin-regulated transport of glucose into muscle and fat cells. This process, which is essential for whole-body glucose homoeostasis in mammals, is thought to be mediated via Akt-dependent movement of GLUT4 glucose transporters to the plasma membrane. In the present study, we have investigated the metabolic side effects of non-ATP-competitive allosteric Akt inhibitors. In 3T3-L1 adipocytes, these inhibitors caused a decrease in the Akt signalling pathway concomitant with reduced glucose uptake. Surprisingly, a similar reduction in GLUT4 translocation to the plasma membrane was not observed. Further investigation revealed that the inhibitory effects of these compounds on glucose uptake in 3T3-L1 adipocytes were independent of the Akt signalling pathway. The inhibitors also inhibited glucose transport into other cell types, including human erythrocytes and T-47D breast cancer cells, suggesting that these effects are not specific to GLUT4. We conclude that these drugs may, at least in part, inhibit tumorigenesis through inhibition of tumour cell glucose transport.

The thioredoxin-thioredoxin reductase system can function in vivo as an alternative system to reduce oxidized glutathione in Saccharomyces cerevisiae.

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E(h)'. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E(h)'. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.

Cu, Zn superoxide dismutase and NADP(H) homeostasis are required for tolerance of endoplasmic reticulum stress in Saccharomyces cerevisiae.

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.