Since the 1950s, one of the goals of adipose tissue research has been to determine lipolytic and lipogenic activity as the primary metabolic pathways affecting adipocyte health and size and thus representing potential therapeutic targets for the treatment of obesity and associated diseases. Nowadays, there is a relatively large number of methods to measure the activity of these pathways and involved enzymes, but their applicability to different biological samples is variable. Here, we review the characteristics of mean lipogenic and lipolytic enzymes, their inhibitors, and available methodologies for assessing their activity, and comment on the advantages and disadvantages of these methodologies and their applicability in vivo, ex vivo, and in vitro, i.e., in cells, organs and their respective extracts, with the emphasis on adipocytes and adipose tissue.

Fasting induces significant shifts in substrate utilization with signs of acute insulin resistance (IR), while obesity is associated with chronic IR. Nonetheless, both states substantially influence adipose tissue (AT) function. Therefore, in this interventional study (NCT04260542), we investigated if excessive adiposity in premenopausal women alters insulin sensitivity and AT metabolic and endocrine activity in response to a 60-h fast and a subsequent 48-h refeeding period. Using physiological methods, lipidomics, and AT explants, we showed that obesity partially modified AT endocrine activity and blunted the dynamics of AT insulin resistance in response to the fasting/refeeding challenge compared to that observed in lean women. AT adapted to its own excess by reducing lipolytic activity/free fatty acids (FFA) flux per mass. This adaptation persisted even after a 60-h fast, resulting in lower ketosis in women with obesity. This could be a protective mechanism that limits the lipotoxic effects of FFA; however, it may ultimately impede desirable weight loss induced by caloric restriction in women with obesity.

Background Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardized protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT’s efficacy and for validating protocol execution. We compared two methods—in vitro cultivation and membrane integrity assessment—for their accuracy, reproducibility, and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Methods Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry; and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Results We refined the bacterial DNA staining protocol integrated with flow cytometry for stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 17%, ranging from 5.3% to 52.9%. Conversely, the membrane integrity approach yielded highly reproducible results, with a median coefficient of variance for viable cells of 0.9%, ranging from 8.5% to 0.04%. Conclusion Bacterial viability assessment using cultivation-dependent methods produces inconsistent outcomes. In contrast, the membrane integrity method offers robust and precise data, making it a viable option for routine faecal material evaluation in FMT.

Objective: Fatty Acid esters of Hydroxy-Fatty Acids (FAHFAs) are anti-diabetic and anti-inflammatory lipokines produced mainly by adipose tissue (AT). As exercise training enhances FAHFA levels, we investigated the impact of acute exercise (AE) and exercise-mimicking conditions on circulating and adipocyte FAHFA levels. Methods: Clinical trial ( NCT05572905 ) in 60 women, grouped by BMI (lean vs. obese) and age (young vs. older), was combined with in vitro experiments on human adipocytes. Following baseline characterization (body composition, VO _2max , insulin sensitivity, AT/plasma FAHFAs), women underwent a cross-over AE and control interventions with repeated blood sampling for FAHFA analysis. Results: In AT, lean and older women exhibited higher FAHFA levels than obese and young women, respectively; older women also showed a shift toward higher levels of 13/12-carbon-branched FAHFAs. Circulating FAHFA levels were similar across all groups and were not positively associated with insulin sensitivity, VO _2max or FAHFA levels in AT. Although AE increased circulating free fatty acids (FFA), plasma FAHFAs dropped in response to both AE and control interventions. In adipocytes, FAHFAs were unaffected by glucocorticoids but increased in response to lipolysis together with gene expression related to FFA oxidation (FAO). Nevertheless, blocking mitochondrial FAO partially mimicked the lipolytic effect, while peroxisomal inhibition synergistically boosted FAHFA lipolysis-driven production despite having no effect alone. Conclusions: While adiposity and aging modulate FAHFA levels in AT, circulating levels remain stable and unaffected by AE, challenging subcutaneous AT as their primary systemic source. In vitro , FAHFA synthesis is driven by high FFA availability but limited by competing peroxisomal FAO.