Metabolic transitions regulate global protein fatty acylation.

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins; however, the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose that influences cellular and mitochondrial uptake/utilization of fatty acids and affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics, and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK, and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.

Intervention by picroside II on FFAs induced lipid accumulation and lipotoxicity in HepG2 cells.

BACKGROUND: Accumulation of free fatty acids (FFAs) in hepatocytes is a hallmark of liver dysfunction and non-alcoholic fatty liver disease (NAFLD). Excessive deposition of FFAs alters lipid metabolism pathways increasing the oxidative stress and mitochondrial dysfunction. Attenuating hepatic lipid accumulation, oxidative stress, and improving mitochondrial function could provide potential targets in preventing progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). Earlier studies with Picrorhiza kurroa extract have shown reduction in hepatic damage and fatty acid infiltration in several experimental models and also clinically in viral hepatitis. Thus, the effect of P. kurroa's phytoactive, picroside II, needed mechanistic investigation in appropriate in vitro liver cell model. OBJECTIVE(S): To study the effect of picroside II on FFAs accumulation, oxidative stress and mitochondrial function with silibinin as a positive control in in vitro NAFLD model. MATERIALS AND METHODS: HepG2 cells were incubated with FFAs-1000µM in presence and absence of Picroside II-10 µM for 20 hours. RESULTS: HepG2 cells incubated with FFAs-1000µM lead to increased lipid accumulation. Picroside II-10µM attenuated FFAs-induced lipid accumulation (33%), loss of mitochondrial membrane potential (ΔΨm), ATP depletion, and production of reactive oxygen species (ROS). A concomitant increase in cytochrome C at transcription and protein levels was observed. An increase in expression of MnSOD, catalase, and higher levels of tGSH and GSH:GSSG ratios underlie the ROS salvaging activity of picroside II. CONCLUSION: Picroside II significantly attenuated FFAs-induced-lipotoxicity. The reduction in ROS, increased antioxidant enzymes, and improvement in mitochondrial function underlie the mechanisms of action of picroside II. These findings suggest a need to develop an investigational drug profile of picroside II for NAFLD as a therapeutic strategy. This could be evaluated through the fast-track path of reverse pharmacology.

Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria.

Anabolic and catabolic signaling mediated via mTOR and AMPK (AMP-activated kinase) have to be intrinsically coupled to mitochondrial functions for maintaining homeostasis and mitigate cellular/organismal stress. Although glutamine is known to activate mTOR, whether and how differential mitochondrial utilization of glutamine impinges on mTOR signaling has been less explored. Mitochondrial SIRT4, which unlike other sirtuins is induced in a fed state, is known to inhibit catabolic signaling/pathways through the AMPK-PGC1α/SIRT1-peroxisome proliferator-activated receptor α (PPARα) axis and negatively regulate glutamine metabolism via the tricarboxylic acid cycle. However, physiological significance of SIRT4 functions during a fed state is still unknown. Here, we establish SIRT4 as key anabolic factor that activates TORC1 signaling and regulates lipogenesis, autophagy, and cell proliferation. Mechanistically, we demonstrate that the ability of SIRT4 to inhibit anaplerotic conversion of glutamine to α-ketoglutarate potentiates TORC1. Interestingly, we also show that mitochondrial glutamine sparing or utilization is critical for differentially regulating TORC1 under fed and fasted conditions. Moreover, we conclusively show that differential expression of SIRT4 during fed and fasted states is vital for coupling mitochondrial energetics and glutamine utilization with anabolic pathways. These significant findings also illustrate that SIRT4 integrates nutrient inputs with mitochondrial retrograde signals to maintain a balance between anabolic and catabolic pathways.

Generation and characterization of human iPSC line from CD34+ cells isolated from umbilical cord blood belonging to Indian origin.

We describe here the reprogramming of CD34+ cells isolated from umbilical cord blood obtained after full term delivery of a healthy female child of Indian origin. The cells were nucleofected by episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). Colonies were identified by alkaline phosphatase staining and characterized for expression of pluripotency markers at protein level by immunofluorescence, flow cytometry and at transcript level by PCR. Genomic stability of the cell line was checked by G-banded karyotype. The ability to differentiate to endoderm, mesoderm and ectoderm in vitro was confirmed by immunofluorescence staining.

Valproic acid enhances the neural differentiation of human placenta derived-mesenchymal stem cells in vitro.

Mesenchymal stem cells (MSCs) are known to express a wide range of markers belonging to all the three lineages: mesodermal, ectodermal and endodermal. Therefore, the possibility of their transdifferentiation towards a neural lineage has been an aspect of active research. In the present study, MSCs were isolated from human placental tissue (P-MSC) and subjected them to neural differentiation. It was found that the P-MSCs differentiated towards neural lineage in appropriate differentiation conditions. However, when a histone deacetylase (HDAC) inhibitor - valproic acid (VPA) - was incorporated in the medium, there was a further increase in their neural differentiation potential. The increase in the number of neurites and neural lineage specific markers was notable. The VPA-treated cells showed a significantly elevated membrane potential compared with the cells grown in only differentiation medium. When the molecular mechanism was studied, the enhancement in the neuronal lineage specification was caused by the inhibition of bone morphogenetic protein (BMP) 2 and an increase in BMP4 under both conditions. The target of VPA (HDAC2) was reduced in the VPA set, whereas HDAC1 remained unchanged. Concurrent reduction in the levels of Stat3 was observed, leading to an upregulation of ßIII tubulin, which is a neuronal lineage-specific marker. The components of Notch signalling (i.e. decreased notch 1 and increased notch 3) also supported differentiation towards the neuronal lineage. Thus, the VPA treated P-MSCs can serve as an alternative source for deriving neural cells for use in both research and in clinics. Copyright © 2016 John Wiley & Sons, Ltd.

Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study.

Mesenchymal stem cells (MSCs) show immunoregulatory properties. Here, we compared MSCs obtained from placenta (P-MSCs) and umbilical cord (C-MSCs) from the same donor, for their immunomodulatory efficacy. P-MSCs and C-MSCs showed similar morphology and phenotypic profile, but different clonogenic ability. Importantly, they showed a significant difference in their immunosuppressive properties as assessed in mixed leukocyte reaction (MLR). The P-MSCs affected the antigen presenting ability of mononuclear cells (MNCs) and dendritic cells (DCs) significantly as compared to C-MSCs resulting in a reduced T-cell proliferation. P-MSC conditioned medium (CM) showed a significant reduction in T cell proliferation as compared to C-MSC CM, thus suggesting that a cell to cell contact is not essential. We found increased levels of IL-10 and TGFß1 and reduction in levels of IFNγ in P-MSC MLRs as compared to C-MSC MLRs. Furthermore, the CD3(+) CD4(+) CD25(+) T regulatory cells were enriched in case of P-MSCs in both, MSC-MNC and MSC-DC co-cultures. This observation was further supported by increased mRNA expression of FoxP3 in P-MSCs. Presently, cord-derived MSCs are being employed in transplantation therapies parallel to the bone marrow-derived MSCs. Our findings suggest that P-MSCs can be a better alternative to C-MSCs, to provide aid in immunological ailments.