The hallmark of white adipocytes is a single, large lipid droplet, yet this unilocular morphology has remained challenging to reproduce in vitro due to limited understanding of its molecular drivers. Here, we identify metabolic reprogramming as the key determinant of adipocyte unilocularity in human 3D cultures. Transcriptomic and metabolomic profiling revealed unilocularity to be primarily characterized by enhanced aerobic glycolysis and reduced mitochondrial content. Importantly, this mirrored the metabolic pattern of freshly isolated human white adipocytes but contrasted with that of preadipocytes and 2D cultures. We demonstrate that aerobic glycolysis in adipocytes activates AMP-activated protein kinase (AMPK), which then enhances CD36-mediated fatty acid uptake. Pharmacological inhibition of aerobic glycolysis reduced fatty acid uptake and led to a more multilocular phenotype, which could be rescued by reactivating AMPK. Importantly, limiting mitochondrial activity or activating AMPK in multilocular 3D cultures was enough to promote their unilocularity. These findings establish aerobic glycolysis as a key driver of white adipocyte lipid droplet morphology and size. They also underscore the influence of cellular microenvironment on shaping adipocyte metabolism and function. Taken together, the study provides important insights of adipocyte lipid droplet biology that can be used to improve human adipocyte functionality and prevent disease.

Oxidized phospholipids (OxPL) are bioactive lipid species that circulate bound to apolipoprotein B-100 [apoB] and apolipoprotein(a) [apo(a)] and have been widely studied as biomarkers of oxidative lipid burden. When bound to apolipoprotein B-100 [OxPL-apoB] and apolipoprotein(a) [OxPL-apo(a)], they serve as informative biomarkers for CVD risk prediction, risk reclassification, and therapeutic monitoring, particularly in studies involving RNA-targeted therapies against lipoprotein(a). To date, measurement of OxPL-apoB and OxPL-apo(a) has been limited to research-use assays performed in an academic laboratory without formal clinical laboratory validation. Here we report the first full CLIA-compliant analytical validation of chemiluminescent ELISA methods for OxPL-apoB and OxPL-apo(a), enabling their implementation in a regulated clinical reference laboratory setting. The OxPL-apoB ELISA employs murine monoclonal IgG antibody MB47 to capture apoB-100-containing lipoproteins, while the OxPL-apo(a) employs murine monoclonal IgG antibody LPA4 to capture apo(a)-containing particles. In both assays, OxPL is detected by murine monoclonal IgM antibody biotin-E06. The concentration of OxPL is determined against a standard curve of phosphocholine (PC) equivalents using PC-modified bovine serum albumin. The analytical measuring range of both assays is 1.48-148.48 nmol/L PC-OxPL. Serum and plasma matrices showed minimal bias and were analytically equivalent. In healthy donors, OxPL-apoB levels ranged from <1.48 to 25.23 nmol/L PC-OxPL (mean 4.18, median 1.79 nmol/L), while OxPL-apo(a) levels ranged from <1.48 to 126.94 nmol/L PC-OxPL (mean 31.04, median 6.90 nmol/L), with strong correlation to Lp(a) concentrations (R2 = 0.82). These assays provide robust tools for quantifying proatherogenic and pro-inflammatory OxPL-lipoprotein complexes in clinical, translational, and pharmacological research settings.

Liquid–liquid phase separation has emerged as a central organizing mechanism that drives the formation of biomolecular condensates and enables cells to spatially and temporally coordinate metabolism, signaling, and gene expression. While the influence of post-translational modifications such as phosphorylation and ubiquitination on condensate behavior is well-established, the contribution of lipidation, the covalent attachment of lipid moieties to proteins, to these processes has received far less attention. Lipidation dictates protein hydrophobicity, membrane affinity, and subcellular distribution, yet how these parameters influence LLPS and thereby modulate condensate dynamics remains unclear. We propose that lipidation operates as a molecular code that integrates membrane association with phase separation, thereby tuning the assembly, composition, and thus functional output of condensates. Extending this concept beyond classical membrane systems, we further suggest that nuclear phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), may act as an unconventional lipid modifier that structures membraneless nuclear compartments through a process termed PIPoylation. Drawing on recent findings, we outline how canonical covalent lipidations, including palmitoylation, myristoylation, prenylation, and phospholipidation, govern membrane nanodomain organization, autophagy, and nuclear condensate architecture. We discuss how covalent lipidation influences condensate wetting, membrane curvature, and lipid–protein demixing, and how PI(4,5)P2 metabolism links chromatin remodeling with transcriptional control via LLPS. Together, these mechanisms underscore lipidation as a crucial regulator of condensate-membrane communication across cellular compartments.

Constitutive photomorphogenesis mutant 9 (COP9) signalosome (CSN) is composed of eight subunits (CSN1 through CSN8). It acts as an essential regulator of Cullin-RING-ubiquitin ligases (CRLs), which target critical cellular regulators for degradation via the ubiquitin (Ub) proteasome pathway. The role of CSN in adipose tissue development and function has not yet been studied. We sought to determine the role of CSN8, the smallest subunit of the CSN complex, in adipogenesis, adipose tissue maintenance, and metabolic balance. We first found that CSN8 level remained constant during adipogenesis and knocking down CSN8 by CRISPR/Cas9 did not impair adipocyte differentiation. Notably, mice with adipocyte-specific Csn8 gene deletion (Csn8AKO) showed disrupted CSN holo-complex formation and Cullin deneddylation, leading to the loss of white and brown adipose tissue. Csn8AKO mice displayed insulin resistance while maintaining glucose tolerance. They showed increased food intake and a trend toward higher energy expenditure but were cold-intolerant. Bulk RNA sequencing revealed that CSN deficiency caused significant remodeling of white and brown adipose tissues, characterized by adipocyte death and inflammation. Specifically, white and brown adipose tissues lacking CSN8 exhibited marked upregulation of apoptotic and pyroptotic cell death, which was associated with alterations in ubiquitination and proteasome activity. In addition, Csn8AKO mice were protected from high-fat diet-induced adipose tissue expansion but developed notable hepatomegaly, steatosis, and insulin resistance. Taken together, our data highlights that CSN8/CSN is crucial for maintaining protein homeostasis in adipose tissue, promoting adipocyte survival, supporting adipose tissue maintenance, and overall metabolic health.

The high-density lipoprotein (HDL) is the most heterogeneous and protein-rich lipoprotein, and its complex proteome has been correlated with many distinct properties. However, the lack of a standardized approach for HDL isolation, combined with the fact that different methods capture overlapping, but distinct HDL subspecies make the establishment of a composition-function relationship a challenge. Key factors influencing HDL proteomic profile are the isolation methodology and the technical variability associated with it. Importantly, interlaboratory technical variability associated with HDL isolation methodologies and how it affects the HDL proteome has never been determined. Here, we used two common methods to isolate HDL particles, ultracentrifugation (UC) and immunoaffinity chromatography against APOA1 (IAC), and performed a thorough evaluation of intralaboratory repeatability as well as intra- and interlaboratory reproducibility of these isolation methods, assessing their influence on HDL-associated proteins composition and abundance. Our results demonstrate that methodological variability has a greater impact on the HDL proteome than interlaboratory differences, with almost 60% of the variance in the data explained by the method of isolation. Importantly, the top 15 HDL proteins account for > 90% of the protein mass in HDL, regardless of the isolation method, and the variability in protein quantification is inversely associated with protein abundance. A joint analysis combining interlaboratory reproducibility of top 15 HDL proteins in multiple days shows 11 and 10 proteins with CV < 25% for UC and IAC, respectively. In summary, our findings demonstrate that standardized isolation methods can achieve acceptable reproducibility within and across laboratories, but they may capture distinct HDL particles.

The mammary gland synthesizes and secretes nutrient-rich milk containing lactose, protein, and lipids, with the complex assortment of lipids providing more than half of the energy and bioactive factors that impact the growth and development of neonates. The birth of neonates initiates the lipogenic capacity of the mammary gland with upregulation in expression of lipogenic genes, including Stearoyl-CoA desaturase (Scd1). SCD1 plays a critical role in lipogenesis, catalyzing the conversion of saturated fatty acids to monounsaturated fatty acids. Previous studies of Scd1 knockout mice revealed that SCD1 impacts several metabolic processes in the liver and adipose tissue, including fat synthesis. However, the role of SCD1 in lactation is not fully understood. Our study aimed to determine the role of SCD1 in lactation and the effects of maternal knockout of Scd1 on the growth of the lactating neonates. We employed second-parity Scd1-deficient female mice (n = 7) that we compared with wild-type mice (n = 6). To determine lipid and metabolic alterations, mammary gland and milk samples were harvested on lactation day 10. Relative to wild-type mice, mammary gland weight, alveolar area, and milk glycerolipid content were reduced in lactating Scd1-deficient mice. Scd1 deficiency also diminished mammary gland biosynthetic metabolic pathways, such as glycerolipid and phospholipid synthesis, while enhancing catabolic pathways, such as fatty acid oxidation. Neonates nursed by Scd1-deficient mice exhibited lower body weights. These findings highlight the critical role of SCD1 in orchestrating metabolic adaptations during lactation to ensure adequate milk synthesis to support the rapidly growing neonates.