Yuen-Li Chung

SREBP Activity Is Regulated by mTORC1 and Contributes to Akt-Dependent Cell Growth

Summary Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.

PKB/Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP.

Protein kinase B (PKB/Akt) has been shown to play a role in protection from apoptosis, cell proliferation and cell growth. It is also involved in mediating the effects of insulin, such as lipogenesis, glucose uptake and conversion of glucose into fatty acids and cholesterol. Sterol-regulatory element binding proteins (SREBPs) are the major transcription factors that regulate genes involved in fatty acid and cholesterol synthesis. It has been postulated that constitutive activation of the phosphatidylinositol 3 kinase/Akt pathway may be involved in fatty acid and cholesterol accumulation that has been described in several tumour types. In this study, we have analysed changes in gene expression in response to Akt activation using DNA microarrays. We identified several enzymes involved in fatty acid and cholesterol synthesis as targets for Akt-regulated transcription. Expression of these enzymes has previously been shown to be regulated by the SREBP family of transcription factors. Activation of Akt induces synthesis of full-length SREBP-1 and SREBP-2 proteins as well as expression of fatty acid synthase (FAS), the key regulatory enzyme in lipid biosynthesis. We also show that Akt leads to the accumulation of nuclear SREBP-1 but not SREBP-2, and that activation of SREBP is required for Akt-induced activation of the FAS promoter. Finally, activation of Akt induces an increase in the concentration of cellular fatty acids as well as phosphoglycerides, the components of cellular membranes. Our data indicate that activation of SREBP by Akt leads to the induction of key enzymes of the cholesterol and fatty acid biosynthesis pathways, and thus membrane lipid biosynthesis.

Proteomic and Metabolomic Analyses of Atherosclerotic Vessels From Apolipoprotein E-Deficient Mice Reveal Alterations in Inflammation, Oxidative Stress, and Energy Metabolism

Objective—Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E−/− mice on normal chow diet. Methods and Results—Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E−/− mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E−/− mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E−/− mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool. Conclusion—Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism.

Combined metabolomic and proteomic analysis of human atrial fibrillation.

OBJECTIVES We sought to decipher metabolic processes servicing the increased energy demand during persistent atrial fibrillation (AF) and to ascertain whether metabolic derangements might instigate this arrhythmia. BACKGROUND Whereas electrical, structural, and contractile remodeling processes are well-recognized contributors to the self-perpetuating nature of AF, the impact of cardiac metabolism upon the persistence/initiation of this resilient arrhythmia has not been explored in detail. METHODS Human atrial appendage tissues from matched cohorts in sinus rhythm (SR), from those who developed AF post-operatively, and from patients in persistent AF undergoing cardiac surgery were analyzed using a combined metabolomic and proteomic approach. RESULTS High-resolution proton nuclear magnetic resonance (NMR) spectroscopy of cardiac tissue from patients in persistent AF revealed a rise in beta-hydroxybutyrate, the major substrate in ketone body metabolism, along with an increase in ketogenic amino acids and glycine. These metabolomic findings were substantiated by proteomic experiments demonstrating differential expression of 3-oxoacid transferase, the key enzyme for ketolytic energy production. Notably, compared with the SR cohort, the group susceptible to post-operative AF showed a discordant regulation of energy metabolites. Combined principal component and linear discriminant analyses of metabolic profiles from proton NMR spectroscopy correctly classified more than 80% of patients at risk of AF at the time of coronary artery bypass grafting. CONCLUSIONS The present study characterized the metabolic adaptation to persistent AF, unraveling a potential role for ketone bodies, and demonstrated that discordant metabolic alterations are evident in individuals susceptible to post-operative AF.

Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models

MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors.

Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism

Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.

Model free approach to kinetic analysis of real-time hyperpolarized 13C magnetic resonance spectroscopy data.

Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized 13C metabolic imaging in humans, where measurement of the input function can be problematic.

Glut-1 as a therapeutic target: increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies

The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. “Positive” COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis (31P-MRS and 1H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.

Proteomic and metabolomic analysis of atrial profibrillatory remodelling in congestive heart failure

Congestive heart failure (CHF) leads to atrial structural remodelling and increased susceptibility to atrial fibrillation. The underlying molecular mechanisms are poorly understood. We applied high-throughput proteomic and metabolomic analysis to left-atrial cardiomyocytes and tissues obtained from sham and ventricular-tachypaced (VTP, 240 bpm × 24 h and × 2 weeks) CHF dogs. Protein-extracts were subjected to two-dimensional gel electrophoresis using differential in-gel electrophoresis technology. Differentially expressed (P<0.05) proteins were identified by tandem mass-spectrometry. Cardiac metabolites were assayed with high-resolution NMR spectroscopy. Extensive changes occurred in structural proteins, particularly at 2-week VTP, with desmin and filamin fragmentation suggesting structural damage, which was confirmed by electron-microscopy. Oxidant stress was evidenced by decreased antioxidant proteins (superoxide dismutase and peroxiredoxin) at 2-week VTP. Extensive changes in cardioprotective heat shock proteins (HSPs) occurred, with several proteins increasing rapidly (HSP27, HSP60 and HSP70) and others showing a delayed rise (GRP78, α-B-crystallin, and HSP90). An evolving adaptive response to metabolic stress was suggested by early upregulation of malate dehydrogenase (DH), α-/β-enolase and pyruvate dehydrogenase (α-subunit of E1 component) and delayed downregulation of a host of enzymes, along with extensive metabolomic changes. Early changes in metabolite expression that persisted as CHF developed included increased concentrations of glucose and alanine. ADP/ATP accumulation and alpha-ketoisovalerate depletion at 2-week VTP suggested a combination of metabolic stress and less effective energy utilization, as well as a shift from glycolysis to alpha-ketoacid metabolism. We conclude that VTP-induced CHF causes time-dependent changes in the atrial proteome and metabolome, providing insights into molecular mechanisms contributing to arrhythmogenic atrial remodelling.

Proteomic and Metabolomic Analysis of Smooth Muscle Cells Derived From the Arterial Media and Adventitial Progenitors of Apolipoprotein E–Deficient Mice

We have recently demonstrated that stem cell antigen 1–positive (Sca-1+) progenitors exist in the vascular adventitia of apolipoprotein E–deficient (apoE−/−) mice and contribute to smooth muscle cell (SMC) accumulation in vein graft atherosclerosis. Using a combined proteomic and metabolomic approach, we now characterize these local progenitors, which participate in the formation of native atherosclerotic lesions in chow-fed apoE−/− mice. Unlike Sca-1+ progenitors from embryonic stem cells, the resident Sca-1+ stem cell population from the vasculature acquired a mature aortic SMC phenotype after platelet-derived growth factor-BB stimulation. It shared proteomic and metabolomic characteristics of apoE−/− SMCs, which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the differentially expressed proteins were key enzymes in glucose metabolism, resulting in faster glucose consumption and a compensatory reduction in baseline interleukin-6 secretion. The latter was associated with a marked upregulation of insulin-like growth factor binding proteins (IGFBPs) 3 and 6. Notably, reconstitution of interleukin-6 to levels measured in the conditioned medium of wild-type SMCs attenuated the elevated IGFBP expression in apoE−/− SMCs and their vascular progenitors. This coregulation of apoE, interleukin-6, and IGFBPs was replicated in wild-type SMCs from hypercholesterolemic mice and confirmed by silencing apoE expression in SMCs from normocholesterolemic mice. In summary, we provide evidence that Sca-1+ progenitors contribute to native atherosclerosis in apoE−/− mice, that apoE deficiency and hypercholesterolemia alter progenitor cell behavior, and that inflammatory cytokines such as interleukin-6 act as metabolic regulators in SMCs of hyperlipidemic mice.