Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

A quantitative pipeline to assess secretion of human leptin coding variants reveals mechanisms underlying leptin deficiencies.

The hormone leptin, primarily secreted by adipocytes, plays a crucial role in regulating whole-body energy homeostasis. Homozygous loss-of-function mutations in the leptin gene (LEP) cause hyperphagia and severe obesity, primarily through alterations in leptin's affinity for its receptor or changes in serum leptin concentrations. Although serum concentrations are influenced by various factors (e.g., gene expression, protein synthesis, stability in the serum), proper delivery of leptin from its site of synthesis in the endoplasmic reticulum via the secretory pathway to the extracellular serum is a critical step. However, the regulatory mechanisms and specific machinery involved in this trafficking route, particularly in the context of human LEP mutations, remain largely unexplored. We have employed the Retention Using Selective Hooks system to elucidate the secretory pathway of leptin. We have refined this system into a medium-throughput assay for examining the pathophysiology of a range of obesity-associated LEP variants. Our results reveal that leptin follows the default secretory pathway, with no additional regulatory steps identified prior to secretion. Through screening of leptin variants, we identified three mutations that lead to proteasomal degradation of leptin and one variant that significantly decreased leptin secretion, likely through aberrant disulfide bond formation. These observations have identified novel pathogenic effects of leptin variants, which can be informative for therapeutics and diagnostics. Finally, our novel quantitative screening platform can be adapted for other secreted proteins.

Moderate-intensity constant or high-intensity interval training? Metabolic effects on candidates to undergo bariatric surgery.

BACKGROUND & AIMS: Bariatric surgery is highly effective against obesity. Pre-surgical exercise programs are recommended to prepare the candidate physically and metabolically for surgery-related rapid weight loss. However, the ideal exercise prescription in this population is unknown. This study aimed to compare the metabolic effects of moderate-intensity constant (MICT) vs. a high-intensity interval training (HIIT) program in candidates to undergo bariatric surgery. METHODS AND RESULTS: Twenty-five candidates (22 women) to undergo sleeve gastrectomy aged from 18 to 60 years old were recruited. At baseline, we measured body composition, physical activity levels, grip strength, and aerobic capacity. Further, we assessed metabolic function through glycemia and insulinemia (both fasting and after oral glucose tolerance test (OGTT)), homeostatic model assessment for insulin resistance (HOMA-IR), lipid profile, glycated haemoglobin (HbA1c), transaminases, fibroblast growth factor 21 (FGF21), growth differentiation factor 15 (GDF15), apelin, and adiponectin. Afterward, participants were randomized into MICT (n = 14) or HIIT (n = 11). Both training programs consisted of 10 sessions (2-3 times/week, 30 min per session) distributed during 4 weeks before the surgery. After this, all outcomes were measured again at the end of the training programs and 1 month after the surgery (follow-up). A mixed effect with Tukey's post-hoc analysis was performed to compare values at baseline vs. post-training vs. postsurgical follow-up. Both training programs increased aerobic capacity after training (p < 0.05), but only after MICT these changes were kept at follow-up (p < 0.05). However, only MICT decreased fat mass and increased total muscle mass and physical activity levels (p < 0.05). Metabolically, MICT decreased insulinemia after OGTT (p < 0.05), whereas HIIT increased adiponectin after training and GDF15 at follow-up (both p < 0.05). CONCLUSIONS: Both MICT and HIIT conferred benefits in candidates to undergo bariatric surgery, however, several of those effects were program-specific, suggesting that exercise intensity should be considered when preparing these patients. Future studies should explore the potential benefits of prescribing MICT or HIIT in a customized fashion depending on a pretraining screening, along with possible summatory effects by combining these two exercise programs (MICT + HIIT). CLINICAL TRIAL REGISTRATION: International Traditional Medicine Clinical Trial Registry, N° ISRCTN42273422.

Moderate-Intensity Constant and High-Intensity Interval Training Confer Differential Metabolic Benefits in Skeletal Muscle, White Adipose Tissue, and Liver of Candidates to Undergo Bariatric Surgery.

Background/Objectives: Bariatric surgery candidates require presurgical physical training, therefore, we compared the metabolic effects of a constant moderate-intensity training program (MICT) vs. a high-intensity interval training (HIIT) in this population. Methods: Seventeen participants performed MICT (n = 9, intensity of 50% of heart rate reserve (HRR) and/or 4-5/10 subjective sensation of effort (SSE)) or HIIT (n = 8, 6 cycles of 2.5 min at 80% of the HRR and/or 7-8/10 of SSE, interspersed by 6 cycles of active rest at 20% of the FCR) for 10 sessions for 4 weeks. After training, tissue samples (skeletal muscle, adipose tissue, and liver) were extracted, and protein levels of adiponectin, GLUT4, PGC1α, phospho-AMPK/AMPK, collagen 1 and TGFß1 were measured. Results: Participants who performed MICT showed higher protein levels of PGC-1α in skeletal muscle samples (1.1 ± 0.27 vs. 0.7 ± 0.4-fold change, p < 0.05). In the liver samples of the people who performed HIIT, lower protein levels of phospho-AMPK/AMPK (1.0 ± 0.37 vs. 0.52 ± 0.22-fold change), PGC-1α (1.0 ± 0.18 vs. 0.69 ± 0.15-fold change), and collagen 1 (1.0 ± 0.26 vs. 0.59 ± 0.28-fold change) were observed (all p < 0.05). In subcutaneous adipose tissue, higher adiponectin levels were found only after HIIT training (1.1 ± 0.48 vs. 1.9 ± 0.69-fold change, p < 0.05). Conclusions: Our results show that both MICT and HIIT confer metabolic benefits in candidates undergoing bariatric surgery; however, most of these benefits have a program-specific fashion. Future studies should aim to elucidate the mechanisms behind these differences.