Protocol to generate human liver spheroids to study liver fibrosis induced by metabolic stress.

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies.

Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

Tracing the Diverse Paths of One-Carbon Metabolism in Cancer and Beyond.

One-carbon (1C) metabolism is a network of biochemical reactions distributed across organelles that delivers folate-activated 1C units to support macromolecule synthesis, methylation, and reductive homeostasis. Fluxes through these pathways are up-regulated in highly proliferative cancer cells, and anti-folates, which target enzymes within the 1C pathway, have long been used in the treatment of cancer. In this work, we review fundamental aspects of 1C metabolism and place it in context with other biosynthetic and redox pathways, such that 1C metabolism acts to bridge pathways across compartments. We further discuss the importance of stable-isotope-tracing techniques combined with mass spectrometry analysis to study 1C metabolism and conclude by highlighting therapeutic approaches that could exploit cancer cells' dependency on 1C metabolism.

EGFR-driven lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

Long chain monomethyl branched-chain fatty acid levels in human milk vary with gestational weight gain.

Breastfeeding is an important determinant of infant health and there is immense interest in understanding its metabolite composition so that key beneficial components can be identified. The aim of this research was to measure the fatty acid composition of human milk in an Irish cohort where we examined changes depending on lactation stage and gestational weight gain trajectory. Utilizing a chromatography approach optimal for isomer separation, we identified 44 individual fatty acid species via GCMS and showed that monomethyl branched-chain fatty acids(mmBCFA's), C15:0 and C16:1 are lower in women with excess gestational weight gain versus low gestational weight gain. To further explore the potential contribution of the activity of endogenous metabolic pathways to levels of these fatty acids in milk, we administered D2O to C57BL/6J dams fed a purified lard based high fat diet (HFD) or low-fat diet during gestation and quantified the total and de novo synthesized levels of fatty acids in their milk. We found that de novo synthesis over three days can account for between 10 and 50 % of mmBCFAs in milk from dams on the low-fat diet dependent on the branched-chain fatty acid species. However, HFD fed mice had significantly decreased de novo synthesized fatty acids in milk resulting in lower total mmBCFAs and medium chain fatty acid levels. Overall, our findings highlight the diverse fatty acid composition of human milk and that human milk mmBCFA levels differ between gestational weight gain phenotypes. In addition, our data indicates that de novo synthesis contributes to mmBCFA levels in mice milk and thus may also be a contributory factor to mmBCFA levels in human milk. Given emerging data indicating mmBCFAs may be beneficial components of milk, this study contributes to our knowledge around the phenotypic factors that may impact their levels.

EGFR-Driven Lung Adenocarcinomas Co-opt Alveolar Macrophage Metabolism and Function to Support EGFR Signaling and Growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation, which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy-resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth. SIGNIFICANCE: Alternate strategies harnessing anticancer innate immunity are required for lung cancers with poor response rates to T cell-based immunotherapies. This study identifies a targetable, mutually supportive, metabolic relationship between macrophages and transformed epithelium, which is exploited by tumors to obtain metabolic and immunologic support to sustain proliferation and oncogenic signaling.