Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver condition worldwide. There is an urgent need to develop new biomarkers to assess disease severity and to define patients with a progressive phenotype. Activin A is a new promising biomarker with conflicting results about liver fibrosis. In this study we investigate levels of Activin A in patients with biopsy proven MASLD. We assess levels of Activin A in regard to fibrosis stage and genetic variant I148M in the patatin-like phospholipase domain-containing protein 3 (PNPLA3). Methods: Activin A levels were assessed in plasma samples from patients with biopsy-proven MASLD in a cross-sectional study. All patients were clinically evaluated and the PNPLA3 I148M genotype of the cohort was assessed. Findings41 patients were included and 27% of these had advanced fibrosis. In MASLD patients with advanced fibrosis, Activin A levels was higher (p < 0.001) and could classify advanced fibrosis with an AUROC for activin A of 0.836 (p < 0.001). Patients homozygous for PNPLA3 I148M G/G had higher levels of activin A than non-homozygotes (p = 0.027). Conclusions: Circulating activin A levels were associated with advanced fibrosis and could be a potential blood biomarker for identifying advanced fibrosis in MASLD. Patients with the risk genotype PNPLA3 I148M G/G had higher levels of activin A proposing activin A as a contributor of the transition from simple steatosis to a fibrotic phenotype.

Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we perform in-depth measurement of metabolism in intact human liver tissue ex vivo using global C-13 tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Isotope tracing allowed qualitative assessment of a wide range of metabolic pathways within a single experiment, confirming well-known features of liver metabolism but also revealing unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Glucose production ex vivo correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes, and could be suppressed by postprandial levels of nutrients and insulin, and also by pharmacological inhibition of glycogen utilization. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.

Adipocyte cellular signaling, normally and in type 2 diabetes, is far from fully studied. We have earlier developed detailed dynamic mathematical models for some well-studied, and partially overlapping, signaling pathways in adipocytes. Still, these models only cover a fraction of the total cellular response. For a broader coverage of the response, large-scale phosphoproteomic data is key. There exists such data for the insulin response of adipocytes, as well as prior knowledge on possible protein-protein interactions associated with a confidence level. However, methods to combine detailed dynamic models with large-scale data, using information about the confidence of included interactions, are lacking. In our new method, we first establish a core model by connecting our partially overlapping models of adipocyte cellular signaling with focus on: 1) lipolysis and fatty acid release, 2) glucose uptake, and 3) the release of adiponectin. We use the phosphoproteome data and prior knowledge to identify phosphosites adjacent to the core model, and then try to add the adjacent phosphosites to the model. The additions of the adjacent phosphosites is tested in a parallel, pairwise approach with low computation time. We then iteratively collect the accepted additions into a layer, and use the newly added layer to find new adjacent phosphosites. We find that the first 15 layers (60 added phosphosites) with the highest confidence can correctly predict independent inhibitor-data (70-90 % correct), and that this ability decrease when we add layers of decreasing confidence. In total, 60 layers (3926 phosphosites) can be added to the model and still keep predictive ability. Finally, we use the comprehensive adipocyte model to simulate systems-wide alterations in adipocytes in type 2 diabetes. This new method provide a tool to create large models that keeps track of varying confidence.Competing Interest StatementThe authors have declared no competing interest.

Lipolysis and the release of fatty acids to supply energy fuel to other organs, such as between meals, during exercise, and starvation, are fundamental functions of the adipose tissue. The intracellular lipolytic pathway in adipocytes is activated by adrenaline and noradrenaline, and inhibited by insulin. Circulating fatty acids are elevated in type 2 diabetic individuals. The mechanisms behind this elevation are not fully known, and to increase the knowledge a link between the systemic circulation and intracellular lipolysis is key. However, data on lipolysis and knowledge from in vitro systems have not been linked to corresponding in vivo data and knowledge in vivo. Here, we use mathematical modelling to provide such a link. We examine mechanisms of insulin action by combining in vivo and in vitro data into an integrated mathematical model that can explain all data. Furthermore, the model can describe independent data not used for training the model. We show the usefulness of the model by simulating new and more challenging experimental setups in silico, e.g. the extracellular concentration of fatty acids during an insulin clamp, and the difference in such simulations between individuals with and without type 2 diabetes. Our work provides a new platform for model-based analysis of adipose tissue lipolysis, under both non-diabetic and type 2 diabetic conditions.

The prevalence of obesity has over the last 40 years nearly tripled and obesity is one of the major risk factors of developing type 2 diabetes. Type 2 diabetes was formerly called adultonset diabetes but today, probably due to the rise in childhood obesity, it is also seen in children and adolescents. Type 2 diabetes is diagnosed when the body no longer can control the glucose levels in the blood. This is due to an insulin resistant state in the insulin responding tissues, liver, adipose and muscle and insufficient production of insulin in the pancreas. However, in spite of extensive research the mechanisms behind insulin resistance is still not known.

The adipose tissue is believed to play a major role in the development of whole body insulin resistance. Adipocytes are the most important sites for storage of the high energy containing triacylglycerols. Insulin stimulation causes the adipocyte to increase the uptake of glucose and to reduce lipolysis: the hydrolysis of triacylglycerol and release of glycerol and fatty acids. The insulin signalling network is complex with numerous proteins involved. These signaling proteins not only transmit the insulin signal but also create negative and positive feedbackloops and induce cross talk between different parts of the network and with the signalling of other hormones. One important positive feedback in insulin signalling is the mTORC1 mediated feedback to phosphorylation of IRS1 at serine 307. In paper I we found that in human adipocytes this feedback is not likely catalysed by the assumed kinase S6K1. However we find an immunoprecipitate of mTOR to contain a ser307 phosphorylating kinase.

Scaffolding proteins serve as docking sites for several proteins to promote protein-protein interactions that facilitate signal transduction. In paper II we demonstrate the existence of the scaffolding protein IQGAP1 in human adipocytes and that the expression of IQGAP1 is downregulated in type 2 diabetes. We reveal that IQGAP1 co-localises with caveolae, invaginations of the plasma membrane where the insulin receptor is situated, and that this interaction is increased upon insulin stimulation.

In paper III we focus on the control of lipolysis, and sought to understand the interplay between insulin and beta-adrenergic stimulation. We demonstrated that the re-esterification of fatty acids is downregulated in type 2 diabetes causing an increased release of fatty acids from the cells. We showed that beta-adrenergic stimulation with isoproterenol induced a negative feedback via PKA/Epac1 -> PI3K -> PKB -> PDE3B that reduced the cAMP levels and thereby also reduced lipolysis. We also showed that insulin, in addition to its well-known anti-lipolytic effect, at high concentrations had a positive effect on lipolysis. In conclusion we reveal an intricate control of the stimulation as well as the inhibition of lipolysis induced by both isoproterenol and insulin.

Leukotriene C-4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (540), 5-lipoxygenase activating protein (FLAP) and leukotriene C-4 synthase (LTC4S) participate in its biosynthesis. We report evidence from in situ hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45(-) cells and CD45(+) hematopoietic cells. The latter were further separated into immature (Lin(-)) and mature (Lin(+)) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45(+) cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.

Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reactions catalyzed by 5-lipoxygenase and either leukotriene A4 hydrolase or leukotriene C4 synthase. 5-lipoxygenase activating protein (FLAP) is also required. We have previously reported expression of FLAP in the hematopoietic compartment of the fetal liver raising questions regarding the role of leukotrienes in hematopoietic regulation. Here we report evidence from in situ hybridization, immunohistochemistry and qRT-PCR experiments that the complete LT biosynthesis machinery is abundantly expressed in hematopoietic cells of the fetal mouse liver from e11.5 until birth. FACS sorting of hematopoietic cells from e15.5 liver and adult bone marrow into different subpopulations followed by quantitative RT-PCR analysis showed that expression was confined mainly to myeloid cells but also detected in hematopoietic stem and progenitor cells. Analysis of FLAP knockout mice showed that a lack of this gene abolished LT and reduced 5(S)- hydroxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (HETE) production. Furthermore,  decreased relative numbers of B-lymphocytes and increased numbers of T-lymphocytes were observed in peripheral blood and increased numbers of common lymphoid progenitor cells were observed in BM. Taken together these findings suggest that production of LTs can occur in cells of the fetal and adult hematopoietic compartments and that deficiency of the FLAP gene (and leukotrienes) may affect lymphocyte maturation.