Metabolite and transcriptome analysis during fasting suggest a role for the p53-Ddit4 axis in major metabolic tissues

BACKGROUND: Fasting induces specific molecular and metabolic adaptions in most organisms. In biomedical research fasting is used in metabolic studies to synchronize nutritional states of study subjects. Because there is a lack of standardization for this procedure, we need a deeper understanding of the dynamics and the molecular mechanisms in fasting. RESULTS: We investigated the dynamic changes of liver gene expression and serum parameters of mice at several time points during a 48 hour fasting experiment and then focused on the global gene expression changes in epididymal white adipose tissue (WAT) as well as on pathways common to WAT, liver, and skeletal muscle. This approach produced several intriguing insights: (i) rather than a sequential activation of biochemical pathways in fasted liver, as current knowledge dictates, our data indicates a concerted parallel response; (ii) this first characterization of the transcriptome signature of WAT of fasted mice reveals a remarkable activation of components of the transcription apparatus; (iii) most importantly, our bioinformatic analyses indicate p53 as central node in the regulation of fasting in major metabolic tissues; and (iv) forced expression of Ddit4, a fasting-regulated p53 target gene, is sufficient to augment lipolysis in cultured adipocytes. CONCLUSIONS: In summary, this combination of focused and global profiling approaches provides a comprehensive molecular characterization of the processes operating during fasting in mice and suggests a role for p53, and its downstream target Ddit4, as novel components in the transcriptional response to food deprivation

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

Sinoatrial Beat to Beat Variability Assessed by Contraction Strength in Addition to the Interbeat Interval

Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales

Data_Sheet_1_Sinoatrial Beat to Beat Variability Assessed by Contraction Strength in Addition to the Interbeat Interval.CSV

<p>Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales.</p

α/β-Hydrolase Domain Containing Protein 15 (ABHD15) – an Adipogenic Protein Protecting from Apoptosis

<div><p>Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.</p> </div

Abhd15 is a direct and functional PPARγ target gene.

<p><b>A</b>. Genome organization around the Abhd15 transcription start side (TSS) of 3T3-L1 cells during differentiation with ChIP data of peroxisome proliferator-activated receptor gamma (PPARγ) (day 6 and day 10) and CCAAT-enhancer-binding protein alpha (C/EBPα) (day 10) binding, and Pparγ-Retinoid X receptor (RXRα) direct repeat motif analysis. The data suggest putative PPARγ-RXRα binding ~990 bp and ~440 bp upstream of the Abhd15 TSS. <b>B</b>-<b>D</b>. Abhd15 mRNA levels of 3T3-L1 cells upon PPARγ agonist rosiglitazone (Rosi) treatments. Cells were treated with 1 µM Rosi (<b>B</b>) during differentiation, (<b>C</b>) for 12 and 24 hours on day 7 of differentiation, and (<b>D</b>) for 6, 12, and 24 hours before induction of differentiation, all leading to increased Abhd15 expression. <b>E</b>. Abhd15 mRNA expression in Pparγ -/- and Pparγ +/- mouse embryonic fibroblasts (MEFs). Abhd15 is hardly expressed in Pparγ -/- MEFs and can only be further increased upon addition of Rosi (1 µM) in Pparγ +/- MEFs. <b>F</b>. Sequence map of the sequences containing either one (F2 and F3) or two (F1) of the putative PPARγ-RXRα binding sites, evaluated in figure A, used for the luciferase assay. <b>G</b>. The 3 regions of interest located upstream of the Abhd15 gene were cloned into luciferase reporter vectors (named pGL4.21-F1, pGL4.26-F2, pGL4.21-F3) and cotransfected with either Pparγ/Rxrα expressing vectors or an empty vector (pCMX) into Cos7 cells. The luciferase activity of pGL4.21-F1 and pGL4.21-F3, both containing the putative PPARγ-RXRα binding site ~440 bp upstream to the TSS, were significantly increased when compared to pCMX-transfected cells. Addition of Rosi to cells cotransfected with pGL4.21-F1 or pGL44.21-F3 and Pparγ/Rxrα, again significantly increased luciferase activity. Data is presented as mean ± SD from at least three independent experiments. Statistical significance was determined using the two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.</p

Abhd15 expression is required for adipogenesis.

<p><b>A</b>-<b>D</b>. 3T3-L1 cells were infected with lentiviral particles coding for Abhd15 shRNA (Abhd15_sil) or using a non-target shRNA as control (ntc), selected for puromycin resistance, expanded as a mixed population and differentiated. <b>A</b>. Silencing efficiency during adipogenesis of two knock-down lentiviruses against Abhd15, determined by qPCR assay. <b>B</b>. Protein was harvested at day 4 of differentiation of control (ntc) and Abhd15-silenced 3T3-L1 cells (Abhd15_sil1) and subjected to western blotting using the anti-Abhd15 antibody. β-actin served as loading control. Abhd15 protein expression is decreased in Abhd15-silenced 3T3-L1 cells compared to control cells. n=2 C. Silencing of Abhd15 impairs adipogenesis, indicated by the strongly decreased amount of neutral lipids on day 7 of differentiation, stained with Oil red O. <b>D</b>. Stable silencing of Abhd15 in 3T3-L1 cells showed high influences on the expression levels of various important adipogenic genes on day 5 of differentiation (Cebpα, Pparγ, fatty acid binding protein 4 (Fabp4), fatty acid synthase (Fasn)). <b>E</b>. Transient silencing of Abhd15 by electroporation of siRNAs on day 8 of differentiation did not show any effects onto the mRNA levels of adipogenic genes in fully differentiated 3T3-L1 cells (day 10). Data is presented as mean ± SD from at least three independent experiments if not otherwise stated. Statistical significance was determined using the two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.</p

Abhd15 expression is regulated during adipogenesis and decreased by elevated free fatty acid levels.

<p><b>A</b>-<b>B</b>. Abhd15 mRNA expression is increased during adipocyte differentiation of (<b>A</b>) OP 9 cells, mouse embryonic fibroblasts (MEFs), and (<b>B</b>) human Simpson-Golabi-Behmel syndrome (SGBS) cells. <b>C</b>. Abhd15 mRNA is highly expressed in brown and white adipose tissue (BAT and WAT), to a lower extent in liver (Liv), and hardly in skeletal (SM) and cardiac muscle (CM) of wild-type mice in the fed state. <b>D</b>. Abhd15 mRNA expression is decreased in WAT and BAT of genetically obese mice (ob/ob) compared to wild type (wt) mice. <b>E</b>. Mice fed a high fat diet (HFD, 60% calories in fat) show a decreased Abhd15 mRNA expression in WAT already after 3 days, but still after 15 weeks on this diet. Additionally, aging strongly decreases Abhd15 mRNA levels. <b>F</b>. Abhd15 mRNA expression is regulated depending on the nutritional status in mouse tissues. Upon fasting, the expression is decreased in both BAT and WAT. <b>G</b>. Simulated fasting of fully differentiated 3T3-L1 cells (day 7 of differentiation) with IBMX (0.5 mM) and isoproterenol (10 µM) for 2 hours resulted in reduced Abhd15 mRNA expression. <b>H</b>. Treatment of fully differentiated 3T3-L1 cells (day 7 of differentiation) with palmitic acid (100 µM) strongly reduces Abhd15 mRNA expression. Data is presented as mean ± SD from at least three independent experiments. Statistical significance was determined using the two-tailed Student’s t-test. *p<0.05, **p<0.01.</p