Survival response to increased ceramide involves metabolic adaptation through novel regulators of glycolysis and lipolysis

The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart

Feasibility of a Novel Sparse Orthogonal Collimator–Based Preclinical Total Marrow Irradiation for Enhanced Dosimetric Conformality

Total marrow irradiation (TMI) has significantly improved radiation conditioning for hematopoietic cell transplantation in hematologic diseases by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. Recently, preclinical three-dimensional image–guided TMI has been developed to enhance mechanistic understanding of the role of TMI and to support the development of experimental therapeutics. However, a dosimetric comparison between preclinical and clinical TMI reveals that the preclinical TMI treatment lacks the ability to reduce the dose to some of the vital organs that are very close to the skeletal system and thus limits the ability to evaluate radiobiological relevance. To overcome this limit, we introduce a novel Sparse Orthogonal Collimator (SOC)–based TMI and evaluate its ability to enhance dosimetric conformality. The SOC-TMI–based dose modulation technique significantly improves TMI treatment planning by reducing radiation exposures to critical organs that are close to the skeletal system that leads to reducing the gap between clinical and preclinical TMI

Ceramide transfer protein deficiency compromises organelle function and leads to senescence in primary cells.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs

CERT deficiency leads to incipient ER stress in <i>Cert^gt/gt</i> MEFs.

<p>(A) The total ER ceramide levels are slightly decreased while (B) hexosylceramide levels are increased in <i>Cert^gt/gt</i> compared to the control <i>Cert^+/+</i> MEFs. (C) Basal PDI levels are increased in the mutant MEFs. Upon serum starvation the levels of IRE1α and BiP are increased in the mutant cells. (D) The ER was labeled with ER tracker green. The ER in the mutant show altered morphology and altered dynamics (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092142#pone.0092142.s002" target="_blank">videos S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092142#pone.0092142.s003" target="_blank">S2</a>). (E). Metabolic labeling for phospholipids were performed using ³²P-orthophosphoric agent as a substrate. No visible difference in major phospholipids was observed between the wild type control and the mutant cells. (F) qPCR analysis of transcripts for GLUCS and GALCS shows increased transcript levels in P5 <i>Cert^gt/gt</i> compared to the <i>Cert^+/+</i>MEFs.</p

Altered Golgi dynamics in <i>Cert^gt/gt</i> MEFs.

<p>(A) The Golgi architecture seems to be normal as evidenced by immunofluorescence staining using a Golgi marker (GM130) and (B) The ultrastructure of Golgi seems apparently normal although we see a slightly more fragmented pattern of Golgi compared to the wild type. N- denotes the nucleus and the arrows indicated the Golgi cisternae. (C) The MEFs were incubated with DMB-C5-Cer as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092142#s2" target="_blank">materials and methods</a>. While perinuclear concentration of the ceramide is clearly visible in <i>Cert^+/+</i> the <i>Cert^gt/gt</i> show a diffuse staining indicating a lack of transport of ceramide from the ER to the Golgi complex. (D) Recycling of cargo protein cholera toxin B (CTxB, associated with lipid rafts) between the plasma membrane and the Golgi complex is impaired in the mutant MEFs. (E) Quantification of the extent of recovery after photobleaching.</p

Knockdown of CG8093 and CG6277 in the midgut increases whole body TAG, starvation sensitivity in <i>w¹¹¹⁸</i> and <i>dcerk¹</i> flies and knockdown of CG6277 also leads to cardiac defects.

<p>(A). Midgut specific reduction in CG8093 increases whole body TAG level to 325–350% in control <i>w¹¹¹⁸</i> flies and about 275% in <i>dcerk¹</i> mutants. The TAG level of driver is taken as 100%. n = 3, error bars represent standard deviation. (B). Midgut specific reduction in CG6277 increases whole animal TAG to 150% in control <i>w¹¹¹⁸</i> flies and about 275%–300% in <i>dcerk¹</i> mutants. The TAG level of driver is taken as 100%. n = 3, error bars represent standard deviation. (C). Reduction in CG8093 lipase increases the starvation sensitivity of wild type and <i>dcerk¹</i> flies. CG8093 RNAi flies, esgGAL4 driver flies, 8093 RNAi combined to esgGAL4 in wild type and <i>dcerk¹</i> are subjected to starvation and the number of surviving flies is recorded at 6 hr intervals. 200 flies divided into 10 groups for each genotype are used in one experiment. Three independent experiments are performed and the average percentage survival is shown for each time interval. (D). Reduction in CG6277 lipase increases the starvation sensitivity of wild type and <i>dcerk¹</i> flies. CG6277 RNAi flies, esgGAL4 driver flies, 6277 RNAi combined to esgGAL4 in wild type and <i>dcerk¹</i> are subjected to starvation and the number of surviving flies is recorded at 6 hr intervals. (E). Quantification of cardiac function parameters in RNAi knockdown of CG6277 in wild type and <i>dcerk¹</i>. Knockdown results in decreased heart rate in wild type flies and a similar trend in <i>dcerk¹</i>, increased heart period in both backgrounds, longer diastolic interval in control and mutant and no significant change in systolic interval. n = 11–13 for each group. Bar represents standard error of mean and statistical significance was determined using Student's t test; * denotes P< = 0.05-0.01, ** is P< = 0.01-0.001 is and *** denotes P< = 0.001-0.0001.</p

Metabolic reprogramming in <i>dcerk¹</i> mutants.

<p>A. Functional classification of genes whose expression is altered in <i>dcerk¹</i> mutants compared to control <i>w¹¹¹⁸</i><b> flies.</b> RNA extracted from control and mutant flies was hybridized to Affymetrix Drosophila 2.0 microarrays and experiments were carried out in triplicate. Gene expression analysis revealed 152 genes were increased (2-fold or more) and 158 genes were decreased (0.5 fold or less) in <i>dcerk¹</i> compared to <i>w¹¹¹⁸</i>. DAVID gene functional annotation tool and functional classification tool were used to annotate and classify the increased and decreased genes into functionally related gene groups. The top GO terms obtained for increased and decreased genes are listed along with the number of genes altered in each category and the statistical significance based on modified Fisher Exact P-Value. Serine type endopeptidases and hydrolases are upregulated while immune response genes are downregulated in the mutant. <b>B. Metabolomic profiling reveals changes in glycolysis and TCA cycle metabolites in <i>dcerk¹</i> compared to <i>w¹¹¹⁸</i><b>.</b> Control and mutant fly extracts were subjected in triplicate to GC/MS, LC/MS and LC/MS/MS platforms to compare relative levels of several metabolites. The fold change, <i>dcerk¹</i>/<i>w¹¹¹⁸</i> is shown for each metabolite. A p value of <0.05 and a q value of <0.1 (false discovery rate) is considered highly statistically significant (marked by a red *). Among glycolysis intermediates, pyruvate and lactate meet these stringent criteria; pyruvate level is decreased while lactate level is increased in the mutant. Among TCA cycle metabolites, oxaloacetate and α-ketoglutarate are decreased while citrate and malate are increased. Fumarate and succinate are also increased and meet the less stringent statistical criteria of approaching significance (p 0.05 −0.1 and q 0.1 or less, marked by a blue *). Most amino acids (other than cysteine and methionine) and sugars do not show significant differences. Among fatty acids, Dodecanedioate, a long chain fatty acid is decreased significantly in the mutant. This could be due to its degradation to acetyl CoA and succinate, the latter being important for gluconeogenesis during starvation.</b></p

Failure of adaptive mechanisms leads to cardiac dysfunction and reduction in adult life span of <i>dcerk¹</i>.

<p>(A). Measurement of Pglym enzyme activity in young (1 week) and aged (6 weeks) <i>w¹¹¹⁸</i> and <i>dcerk¹</i> flies shows a significant decrease in activity in older <i>dcerk¹</i>. (B). Comparison of transcript levels of CG8093 and CG6277 by QPCR in 1week and 6 week old flies, reveals decrease in their transcripts in aged <i>dcerk¹</i> flies. (C). Comparison of several cardiac performance indices in 1 and 3 week old esgGAL4 <i>dcerk¹</i> flies reveals progressive decline in heart function with decreased heart rate, increased heart period and dilated heart chamber. n = 15, bar represents standard error of mean and statistical significance was determined using Student's t test; * denotes P< = 0.05-0.01, ** is P< = 0.01-0.001 is and *** denotes P< = 0.001-0.0001. (D). Knockdown of CG6277 in 3 week old wild type flies also shows decreased heart rate, increased heart period, dilated heart chamber and arrhythmia. n = 10, bar represents standard error of mean and statistical significance was determined using Student's t test; * denotes P< = 0.05-0.01, ** is P< = 0.01-0.001 is and *** denotes P< = 0.001-0.0001. (E). <i>dcerk¹</i> have reduced adult life span compared to <i>w¹¹¹⁸</i> flies. The survivorship curve of <i>dcerk¹</i> and w<i>¹¹¹⁸</i> represents data from 600 flies in duplicate.</p

Increased glycolytic flux in <i>dcerk¹</i> compared to <i>w¹¹¹⁸</i> due to activation of Pglym and downstream glycolytic enzymes through AKT.

<p>(A). Total AKT and phospho AKT are monitored in control and mutant flies by Western blots. One band is observed for total AKT and it is not significantly different between <i>w¹¹¹⁸</i> and mutant. Two major bands and one minor band are observed for phospho AKT and these are increased in <i>dcerk¹</i> compared to <i>w¹¹¹⁸</i>. The blots also show that total AKT and phospho AKT are both reduced significantly in the hypomorphic allele <i>akt⁴²²⁶</i> used in this study. (B). Quantification of total AKT and phospho AKT levels. Densitometric scanning of total AKT and phospho AKT bands from Western blots was carried out. The phospho AKT graph represents the average obtained after combining the values from all three bands observed on blots. The values were normalized to <i>w¹¹¹⁸</i> taken as 100%. (C). tGPH staining is monitored in control (upper left panel) and <i>dcerk¹</i> (upper right panel) adult midgut. The lower left panel shows tGPH staining in control while the lower right panel shows that in mutant larval fat body. There is increased tGPH staining at the cell membranes (compare arrows) indicating increased PIP3 levels and therefore increased PI3K activity. In addition to plasma membrane, nuclear tGPH is also increased in <i>dcerk¹</i> gut since it has been reported that nuclear tGPH is also visible in many cell types when PIP₃ levels are increased in cells. Midguts from 15–20 flies were visualized in each experiment and three independent experiments were carried out. For visualizing tGPH in fat body, 15–20 early third instar larvae were used in each experiment and three independent experiments were carried out. Scale bar represents 50 µm. (D). FOXO transcript level is measured by QPCR and shows a 60% reduction in in <i>dcerk¹</i> flies compared to <i>w¹¹¹⁸</i>. FOXO transcript level increases in the mutant heads when proteosomal degradation is inhibited by overexpressing DTS5 subunit in <i>dcerk¹</i> photoreceptors compared to <i>w¹¹¹⁸</i> or DTS5 without driver in <i>dcerk¹</i> head samples. (E, F). Transcript level (E) and enzyme activity (F) of Pglym are significantly decreased when AKT activity is genetically reduced in <i>dcerk¹</i> suggesting AKT mediates Pglym activation in <i>dcerk¹</i>. (G). Overexpression of constitutively active FOXO in the muscles (MHC-GAL4) significantly decreases whole body Pglym transcript level. n = 3, error bars represent standard deviation. (H). Reduction of AKT in <i>dcerk¹</i> increases total body trehalose level. Trehalose level is calculated per mg of protein and normalized to <i>w¹¹¹⁸</i> level. n = 3, error bars represent standard deviation. (I). While actin GAL4 driver or UASPglymRNAi transgene individually have no effect on trehalose level, combining them increases whole animal trehalose by 20% in <i>w¹¹¹⁸</i> and 50% in <i>dcerk¹</i>. n = 3, error bars represent standard deviation.</p

Prototype Small-Animal PET-CT Imaging System for Image-Guided Radiation Therapy

Molecular imaging is becoming essential for precision targeted radiation therapy, yet progress is hindered from a lack of integrated imaging and treatment systems. We report the development of a prototype positron emission tomography (PET) scanner integrated into a commercial cone beam computed tomography (CBCT) based small animal irradiation system for molecular-image-guided, targeted external beam radiation therapy. The PET component consists of two rotating Hamamatsu time-of-flight PET modules positioned with a bore diameter of 101.6 mm and a radial field-of-view of 53.1 mm. The measured energy resolution after linearity correction at 511 KeV was 12.9% and the timing resolution was 283.6 ps. The measured spatial resolutions at the field-of-view center and 5 mm off the radial center were 2.6 mm × 2.6 mm × 1.6 mm and 2.6 mm × 2.6 mm × 2.7 mm respectively. 18F-Fluorodeoxyglucose-based PET imaging of a NEMA NU 4-2008 phantom resolved cylindrical volumes with diameters as small as 3 mm. To validate the system in-vivo, we performed 64Cu-DOTA-M5A PET and computed tomography (CT) imaging of carcinoembryonic antigen (CEA)-positive colorectal cancer in athymic nude mice and compared the results with a commercially available Siemens Inveon PET/CT system. The prototype PET system performed comparably to the Siemens system for identifying the location, size, and shape of tumors. Regions of heterogeneous 64Cu-DOTA-M5A uptake were observed. Using 64Cu-DOTA-M5A PET and CT images, a Monte Carlo-based radiation treatment plan was created to escalate the dose to the 64Cu-DOTA-M5A-based, highly active, biological target volume while largely sparing the normal tissue. Results demonstrate the feasibility of molecular-image-guided treatment plans using the prototype theranostic system