In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans.

The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase ALG11 (695-fold) and the phosphatase subunit PPP2CB (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.

Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58(-/-) mice). CGI-58(-/-) macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL(-/-) macrophages. In contrast to ATGL(-/-) macrophages, however, CGI-58(-/-) macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58(-/-) macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE(-/-) mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor(-/-) mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency.

Programmed cell death of lipid-laden macrophages is a prominent feature of atherosclerotic lesions and mostly ascribed to accumulation of excess intracellular cholesterol. The present in vitro study investigated whether intracellular triacylglycerol (TG) accumulation could activate a similar apoptotic response in macrophages. To address this question, we utilized peritoneal macrophages isolated from mice lacking adipose triglyceride lipase (ATGL), the major enzyme responsible for TG hydrolysis in multiple tissues. In Atgl(-/-) macrophages, we observed elevated levels of cytosolic Ca(2+) and reactive oxygen species, stimulated cytochrome c release, and nuclear localization of apoptosis-inducing factor. Fragmented mitochondria prior to cell death were indicative of the mitochondrial apoptosis pathway being triggered as a consequence of defective lipolysis. Other typical markers of apoptosis, such as externalization of phosphatidylserine in the plasma membrane, caspase 3 and poly(ADP-ribose) polymerase cleavage, were increased in Atgl(-/-) macrophages. An artificial increase of cellular TG levels by incubating wild-type macrophages with very low density lipoprotein closely mimicked the apoptotic phenotype observed in Atgl(-/-) macrophages. Results obtained during the present study define a novel pathway linking intracellular TG accumulation to mitochondrial dysfunction and programmed cell death in macrophages.

Fatty acids (FAs) activate and fuel UCP1-mediated non-shivering thermogenesis (NST) in brown adipose tissue (BAT). Release of FAs from intracellular fat stores by adipose triglyceride lipase (ATGL) is considered a key step in NST. Accordingly, the severe cold intolerance of global ATGL knockout (AKO) mice has been attributed to defective BAT lipolysis. Here we show that this conclusion is incorrect. We demonstrate that although the BAT-specific loss of ATGL impairs BAT lipolysis and alters BAT morphology, it does not compromise the β3-adrenergic thermogenic response or cold-induced NST. Instead, NST depends on nutrient supply or lipolysis in white adipose tissue during fasting, suggesting that circulating energy substrates are sufficient to fuel NST. Cold intolerance in AKO mice is not caused by BAT dysfunction as previously suspected but by severe cardiomyopathy. We conclude that functional NST requires adequate substrate supply and cardiac function, but does not depend on ATGL-mediated lipolysis in BAT.

Gut hyperpermeability can be caused by either apoptosis of the intestinal epithelium or altered status, permeability or porosity of tight junctions. This project aims to elucidate these mechanisms in the early phase of sepsis. Eighteen male wild type mice were randomized to two groups. All mice received one single gavage of fluorescein isothiocyanate (FITC) dextran 30 min before intervention. One group (n = 10) underwent cecal ligation and puncture to induce sepsis. The other group (n = 8) was sham operated. Septic animals exhibited significantly increased permeability for FITC 8 h post-operatively. Significantly increased serum interleukin-6, tumor-necrosis-factor-alpha and interleukin-1-beta confirmed sepsis. Septic animals showed significant bowel wall inflammation of ileum and colon samples. PCR revealed significantly increased expression of claudin-2 and decreased expressions of claudin-4, tight-junction-protein-1 and occludin-1 resembling increased permeability of tight junctions. However, these alterations could not be confirmed at the protein level. Light microscopy revealed significant dilatation of intercellular spaces at the basal sections of intestinal epithelial cells (IEC) in septic animals confirmed by increased intercellular spaces at the level of tight junctions and adherens junctions in electron microscopy (TEM). In small angle X-ray scattering no increase in number or size of nanopores could be shown in the bowel wall. HOECHST staining and PCR of ileum samples for apoptosis markers proofed no relevant differences in intestinal epithelial cell apoptosis between the groups. Intestinal hyperpermeability in septic animals was most likely caused by alterations of the intercellular contacts and not by apoptosis or increased size/number of nanopores of intestinal epithelial cells in this murine model of early sepsis.

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis "master-regulator" PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.