Sustained AMPK Activation and Proline Metabolism Play Critical Roles in the Survival of Matrix-Deprived Transformed Cells.

Attachment to the matrix is critical for the survival of adherent cells, whereas detachment triggers death by apoptosis. Therefore, solid tumors must acquire the ability to survive the stress of matrix-detachment to transit through circulation and seed metastases. Although a central role for energy metabolism in cancer progression is well established, what distinguishes its role in the cellular state of the matrix-deprived form compared to the matrix-attached form is not fully understood yet. Using an in vitro transformation model dependent on simian virus 40 (SV40) small t (ST) antigen for cellular survival and proliferation in matrix-deprived conditions, we demonstrate that 5'-adenosine monophosphate-activated protein kinase (AMPK) activity is elevated and sustained under matrix-deprived conditions in ST-expressing fibroblasts. Additionally, these cells display elevated energy (ATP) levels under matrix-deprived conditions in contrast to cells lacking ST expression. The elevated ATP levels are coupled to increased levels of proline in ST-expressing cells, as revealed by metabolomics studies. The AMPK-dependent upregulation of proline oxidase, an enzyme of proline degradation, is a key link for elevated ATP levels. This functional link is further established by proline supplementation concomitant with AMPK activation in matrix-deprived cells lacking ST antigen, yielding ATP and enhancing survival. Thus, our data establishes a key role for AMPK-dependent regulation of proline metabolism in mediating energy homeostasis and promoting survival of matrix-deprived cells. These findings identify key markers that distinguish the metabolic states of matrix-detached and matrix-attached transformed cells and have implications in developing novel therapeutic strategies for specifically targeting matrix-detached metastasizing cancer cells.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart.

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/- mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/- hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1-mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.

Evaluation of Effect of Glucosamine-Chondroitin Sulfate, Tramadol, and Sodium Hyaluronic Acid on Expression of Cytokine Levels in Internal Derangement of Temporomandibular Joint.

AIM: Evaluation of the effect of glucosamine-chondroitin combination, tramadol, and sodium hyaluronic acid in temporomandibular joint (TMJ) disorders and its impact on the expression of various cytokines such as IL-6, IL-1ß, TNF-α, and PGE2. MATERIALS AND METHODS: The present study was conducted on 60 patients (males-30, females-30) suffering from internal derangement such as disc displacement with reduction of TMJ. The patients were divided into three groups of 20 each. Group I received a combination of 1.5g of glucosamine and 1.2 g of chondroitin sulfate per day and group II received 50 mg tramadol HCL peroral. Group III received sodium hyaluronate 10 mg/mL, 2 mL injection syringe on each joint. Pain (VAS) scale and maximum mouth opening (MMO) was measured. The level of IL-6, IL-1ß, TNF-α, and PGE2 levels were measured using Enzyme-linked immuno sorbent assay (ELISA). RESULTS: There was an improvement in maximum mouth opening in all three groups (p < 0.05). There was a reduction in pain in all groups. IL- 1ß, TNF-α, and PGE2 leve ls showed reduction while IL-6 showed an increase in value in group II and III. CONCLUSION: The efficacy of glucosamine chondroitin sulfate , tramadol and hyaluronic acid in TMJ disorders has been found to be effective. CLINICAL SIGNIFICANCE: IL-6, IL-1ß, TNF-α, and PGE2 levels indicate the risk of TMJ disorders. Thus earlier assessment of their levels helps in diagnosis, and better management may be done.

Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum.

Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) [(13)C-(1)H] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a "uniqueness score" and a "coverage score". Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of decluttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMR-based metabolomics data by reducing existing impediments.

A fast NMR method for resonance assignments: application to metabolomics.

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled ((12)C) or (13)C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [(13)C, (1)H] HSQC-TOCSY, (2) 2D (1)H-(1)H TOCSY and (3) 2D (13)C-(1)H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [(13)C, (1)H] HSQC-TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete (1)H and (13)C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.

Amino acid selective labeling and unlabeling for protein resonance assignments.

Structural characterization of proteins by NMR spectroscopy begins with the process of sequence specific resonance assignments in which the (1)H, (13)C and (15)N chemical shifts of all backbone and side-chain nuclei in the polypeptide are assigned. This process requires different isotope labeled forms of the protein together with specific experiments for establishing the sequential connectivity between the neighboring amino acid residues. In the case of spectral overlap, it is useful to identify spin systems corresponding to the different amino acid types selectively. With isotope labeling this can be achieved in two ways: (i) amino acid selective labeling or (ii) amino acid selective 'unlabeling'. This chapter describes both these methods with more emphasis on selective unlabeling describing the various practical aspects. The recent developments involving combinatorial selective labeling and unlabeling are also discussed.