Glucose controls lipolysis through Golgi PtdIns4P-mediated regulation of ATGL.

Metabolic crosstalk of the major nutrients glucose, amino acids and fatty acids (FAs) ensures systemic metabolic homeostasis. The coordination between the supply of glucose and FAs to meet various physiological demands is especially important as improper nutrient levels lead to metabolic disorders, such as diabetes and metabolic dysfunction-associated steatohepatitis (MASH). In response to the oscillations in blood glucose levels, lipolysis is thought to be mainly regulated hormonally to control FA liberation from lipid droplets by insulin, catecholamine and glucagon. However, whether general cell-intrinsic mechanisms exist to directly modulate lipolysis via glucose sensing remains largely unknown. Here we report the identification of such an intrinsic mechanism, which involves Golgi PtdIns4P-mediated regulation of adipose triglyceride lipase (ATGL)-driven lipolysis via intracellular glucose sensing. Mechanistically, depletion of intracellular glucose results in lower Golgi PtdIns4P levels, and thus reduced assembly of the E3 ligase complex CUL7FBXW8 in the Golgi apparatus. Decreased levels of the E3 ligase complex lead to reduced polyubiquitylation of ATGL in the Golgi and enhancement of ATGL-driven lipolysis. This cell-intrinsic mechanism regulates both the pool of intracellular FAs and their extracellular release to meet physiological demands during fasting and glucose deprivation. Moreover, genetic and pharmacological manipulation of the Golgi PtdIns4P-CUL7FBXW8-ATGL axis in mouse models of simple hepatic steatosis and MASH, as well as during ex vivo perfusion of a human steatotic liver graft leads to the amelioration of steatosis, suggesting that this pathway might be a promising target for metabolic dysfunction-associated steatotic liver disease and possibly MASH.

Defatting of Human Livers During Long-Term e x situ Normothermic Perfusion: Novel Strategy to Rescue Discarded Organs for Transplantation.

OBJECTIVE: To develop a protocol for the defatting of steatotic liver grafts during long-term ex situ normothermic machine perfusion. BACKGROUND: Despite the alarming increase in donor organ shortage, the highly prevalent fatty liver grafts are often discarded due to the risk of primary nonfunction. Effective strategies preventing such outcomes are currently lacking. An exciting new avenue is the introduction of ex situ normothermic machine perfusion (NMP), enabling a liver to remain fully functional for up to 2 weeks and providing a unique window of opportunity for defatting before transplantation. METHODS: Over a 5-year period, 23 discarded liver grafts and 28 partial livers from our resection program were tested during ex situ normothermic machine perfusion. The steatosis degree was determined on serial biopsies by expert pathologists, and triglyceride contents were measured simultaneously. RESULTS: Of 51 liver grafts, 20 were steatotic, with up to 85% macrovesicular steatosis, and were perfused for up to 12 days. Ten livers displayed marked (5 of which almost complete) loss of fat, while the other 10 did not respond to long-term perfusion. Successful defatting was related to prolonged perfusion, automated glucose control, circadian nutrition, and L-carnitine/fenofibrate supplementation. Pseudopeliotic steatosis and the associated activation of Kupffer/stellate cells were unexpected processes that might contribute to defatting. Synthetic and metabolic functions remained preserved for most grafts until perfusion ended. CONCLUSION: Ex situ long-term perfusion effectively reduces steatosis while preserving organ viability and may in the future allow transplantation of primarily unusable high-risk grafts, significantly increasing the number of organs available for transplantation.

A Spectrofluorometric Method for Real-Time Graft Assessment and Patient Monitoring.

Biomarkers are powerful clinical diagnostics and predictors of patient outcome. However, robust measurements often require time and expensive laboratory equipment, which is insufficient to track rapid changes and limits direct use in the operating room. Here, this study presents a portable spectrophotometric device for continuous real-time measurements of fluorescent and non-fluorescent biomarkers at the point of care. This study measures the mitochondrial damage biomarker flavin mononucleotide (FMN) in 26 extended criteria human liver grafts undergoing hypothermic oxygenated perfusion to guide clinical graft assessment. Real-time data identified seven organs unsuitable for transplant that are discarded. The remaining grafts are transplanted and FMN values correlated with post-transplant indicators of liver function and patient recovery. Further, this study shows how this device can be used to monitor dialysis patients by measuring creatinine in real-time. Our approach provides a simple method to monitor biomarkers directly within biological fluids to improve organ assessment, patient care, and biomarker discovery.

Observations and findings during the development of a subnormothermic/normothermic long-term ex vivo liver perfusion machine.

BACKGROUND: Ex situliver machine perfusion at subnormothermic/normothermic temperature isincreasingly applied in the field of transplantation to store and evaluateorgans on the machine prior transplantation. Currently, various perfusionconcepts are in clinical and preclinical applications. Over the last 6 years ina multidisciplinary team, a novel blood based perfusion technology wasdeveloped to keep a liver alive and metabolically active outside of the bodyfor at least one week. METHODS: Within thismanuscript, we present and compare three scenarios (Group 1, 2 and 3) we werefacing during our research and development (R&D) process, mainly linked tothe measurement of free hemoglobin and lactate in the blood based perfusate. Apartfrom their proven value in liver viability assessment (ex situ), these twoparameters are also helpful in R&D of a long-term liver perfusion machine and moreover supportive in the biomedical engineering process. RESULTS: Group 1 ("good" liver on the perfusion machine) represents the best liver clearance capacity for lactate and free hemoglobin wehave observed. In contrast to Group 2 ("poor" liver on the perfusion machine), that has shown the worst clearance capacity for free hemoglobin. Astonishingly,also for Group 2, lactate is cleared till the first day of perfusion andafterwards, rising lactate values are detected due to the poor quality of theliver. These two perfusate parametersclearly highlight the impact of the organ quality/viability on the perfusion process. Whereas Group 3 is a perfusion utilizing a blood loop only (without a liver). CONCLUSION: Knowing the feasible ranges (upper- and lower bound) and the courseover time of free hemoglobin and lactate is helpful to evaluate the quality ofthe organ perfusion itself and the maturity of the developed perfusion device. Freehemoglobin in the perfusate is linked to the rate of hemolysis that indicates how optimizing (gentle blood handling, minimizing hemolysis) the perfusion machine actually is. Generally, a reduced lactate clearancecapacity can be an indication for technical problems linked to the blood supplyof the liver and therefore helps to monitor the perfusion experiments.Moreover, the possibility is given to compare, evaluate and optimize developed liverperfusion systems based on the given ranges for these two parameters. Otherresearch groups can compare/quantify their perfusate (blood) parameters withthe ones in this manuscript. The presented data, findings and recommendations willfinally support other researchers in developing their own perfusion machine ormodifying commercially availableperfusion devices according to their needs.

Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR-Cas9.

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. This activated Mg2+-bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.

Tuning the dielectric properties of metallic-nanoparticle/elastomer composites by strain.

Tunable metal/dielectric composites are promising candidates for a large number of potential applications in electronics, sensor technologies and optical devices. Here we systematically investigate the dielectric properties of Ag-nanoparticles embedded in the highly flexible elastomer poly-dimethylsiloxane (PDMS). As tuning parameter we use uniaxial and biaxial strain applied to the composite. We demonstrate that both static variations of the filling factor and applied strain lead to the same behavior, i.e., the filling factor of the composite can be tuned by application of strain. In this way the effective static permittivity εeff of the composite can be varied over a very large range. Once the Poisson's ratio of the composite is known, the strain dependent dielectric constant can be accurately described by effective medium theory without any additional free fit parameter up to metal filling factors close to the percolation threshold. It is demonstrated that, starting above the percolation threshold in the metallic phase, applying strain provides the possibility to cross the percolation threshold into the insulating region. The change of regime from conductive phase down to insulating follows the description given by percolation theory and can be actively controlled.