Ketogenesis requires fatty acid flux from intracellular (lipid droplets) and extrahepatic (adipose tissue) lipid stores to hepatocyte mitochondria. However, whether interorganelle contact sites regulate this process is unknown. Recent studies have revealed a role for Calsyntenin-3β (CLSTN3β), an endoplasmic reticulum-lipid droplet contact site protein, in the control of lipid utilization in adipose tissue. Here, we show that Clstn3b expression is induced in the liver by the nuclear receptor PPARα in settings of high lipid utilization, including fasting and ketogenic diet feeding. Hepatocyte-specific loss of CLSTN3β in mice impairs ketogenesis independent of changes in PPARα activation. Conversely, hepatic overexpression of CLSTN3β promotes ketogenesis in mice. Mechanistically, CLSTN3β affects LD-mitochondria crosstalk, as evidenced by changes in fatty acid oxidation, lipid-dependent mitochondrial respiration, and the mitochondrial integrated stress response. These findings define a function for CLSTN3β-dependent membrane contacts in hepatic lipid utilization and ketogenesis.

Cell death-inducing DFF45-like effector C (CIDEC) condensation through N-terminal phase separation is a prerequisite for CIDEC-mediated lipid droplet (LD) fusion and lipid storage promotion. Here, we present a protocol for in vitro phase separation of the N terminus of CIDEC (CIDEC-N) proteins. We describe steps for plasmid construction, protein expression, and purification. We then detail procedures for concentration detection and in vitro phase separation. This protocol provides a framework to investigate CIDEC-N phase separation properties, laying the foundation for further exploration of its role in lipid metabolism and obesity. For complete details on the use and execution of this protocol, please refer to Lyu et al.1.

Lipid droplets (LDs) are highly dynamic organelles that maintain cellular lipid homeostasis through size and number control. In adipose tissue, CIDEC plays a crucial role in LD fusion and lipid homeostasis. However, the regulatory factors and mechanisms of LD fusion remain largely unknown. Here, we established a high-throughput LD phenotypic screen on a compound library consisting of 2010 small molecules, and identified 11 cytoskeleton inhibitors that negatively regulate LD size. Using specific inhibitors against each of the three types of cytoskeleton, our data showed that the disruption of microtubules and microfilaments but not intermediate filaments limits CIDEC-mediated LD fusion and growth by reducing LD movement and LD-LD contact. The collective effect of microtubule inhibitors results in a small LD phenotype which favors lipolysis upon activation of cAMP-PKA pathway in adipocytes. Our findings demonstrate that cytoskeleton is involved in the process of LD fusion and growth, indicating their role in lipid storage metabolism. One-Sentence Summary: Cytoskeleton regulates lipid droplet fusion and lipid storage.

Context: An adequate supply of energy is essential for the proper functioning of all life activities in living organisms. As organelles that store neutral lipids, lipid droplets (LDs) are involved in the synthesis and metabolism of lipids in cells and are also an important source of energy supply.

Methods and mechanisms: A comprehensive summary of the literature was first carried out to screen for relevant proteins affecting the morphological size of LDs. The size of milk fat globules (MFGs) is directly influenced by the morphological size of LDs, which also controls the energy storage capacity of LDs. In this review, we detail the progress of research into the role of some protein in regulating the morphological size of LDs.

Conclusion: It has been discovered that the number of protein are involved in the control of LD growth and degradation, such as Rab18-mediated local synthesis of triacylglycerol (TAG), cell death-inducing DFF45-like effector family proteins (CIDEs)-mediated atypical fusion between LDs, Stomatin protein-mediated LD fusion and autophagy-related proteins (ATGs)-mediated autophagic degradation of LDs. However, more studies are needed in the future to enrich the network of mechanisms that regulate the morphological size of LDs.

Cold stress poses a significant challenge to pig farming in northern China, leading to reduced productivity and, in severe cases, even mortality. However, the mechanisms underlying cold resistance in pigs are not well understood. To explore the genetic mechanism of cold resistance in pigs under low-temperature conditions, the cold-tolerant Hezuo pig was selected as a model. DIA proteomics analysis was performed on liver tissues from Hezuo pigs after 24 h of exposure to low-temperature treatments. The results showed that approximately 149 differential abundance proteins (DAPs) were detected (95 up-regulated and 54 down-regulated). GO analysis showed that these DAPs were mainly associated with lipid metabolism, vesicle fusion, and membrane function. KEGG analysis showed that these DAPs were primarily enriched in lipid metabolism-related pathways such as cholesterol metabolism and vitamin digestion and absorption. Comprehensive analysis identified APOA4, APOA2, SREBF2, ATP23, STX2, USO1, ETFA, RAB11FIP1, ETNPPL, and SGMS1 as potential key proteins involved in cold resistance mechanisms. The mRNA expression of the genes for two key candidate proteins (APOA4 and SREBF2), which are involved in lipid metabolism, was analyzed using qRT-PCR, revealing a significant up-regulation after low-temperature treatment. These findings provide significant insights into the mechanisms of cold resistance in animals and may serve as candidate markers for further studies on cold tolerance. SIGNIFICANCE: Cold resistance is one of the key traits in pigs and involves multiple complex coordinated regulatory mechanisms. However, its genetic mechanisms are not completely understood. In this study, a DIA proteomics approach was used to identify proteins and pathways associated with cold resistance in the liver of low-temperature-treated Hezuo pigs. These findings offer novel candidate proteins and key pathways for investigating the molecular mechanisms of cold resistance in Hezuo pigs, providing a base for further elucidating the mechanisms of cold tolerance in pigs.

Phosphatidylcholine cytidine transferase α (CCTα) is a key rate-limiting enzyme in the CDP-choline pathway, the primary pathway for phosphatidylcholine (PC) synthesis in mammals. This study investigated the role of CCTα in lipid droplet (LD) formation, phospholipid synthesis, LD fusion, and lipophagy in bovine mammary epithelial cells (BMECs) through CCTα gene knockout (CCT-KO) and overexpression (CCT-OE). CCTα mRNA expression was significantly increased in bovine mammary gland tissue after lactation. In BMECs, CCTα was transferred from the nucleus to the endoplasmic reticulum and localized on LD surfaces in the presence of linoleic acid. Compared with normal BMECs (NC), CCTα knockout (CCT-KO) cells had significantly greater LD diameters (1.53 μm vs. 1.68 μm, p < 0.05), lower proportions of small LDs (<1 µm; 11.39% vs. 5.42%), and higher proportions of large LDs (>3 µm; 0.67% vs. 2.88%). In contrast, CCTα overexpression (CCT-OE) decreased the diameter of LDs to 1.18 μm (p < 0.01), increased the proportion of small LDs to 35.48%, and decreased the proportion of large LDs to 0.24%. CCTα knockout significantly decreased the PC content and the ratio of PC to PE, whereas CCTα overexpression increased the PC content and the ratio of PC to phosphatidyl ethanolamine (PE) (p < 0.05). The lipidomics analysis indicated that PC synthesis was significantly influenced by CCTα gene expression. Live cell observations showed that CCTα knockout promoted the fusion of small LDs into large LDs. In cells with CCT α overexpression, the expression of the microtubule-associated protein 1 light chain 3 (LC3) protein and the number of lysosomes was elevated, and the lysosomal phagocytosis of LDs was observed through transmission electron microscopy, thus indicating that CCTα overexpression enhanced lipophagy. In conclusion, these results suggest that CCTα plays a role in regulating LD formation by influencing PC synthesis, LD fusion, and lipophagy in BMECs.

Membrane contact sites (MCS) are specialized regions where organelles are closely interconnected through membrane structures, facilitating the transfer and exchange of ions, lipids, and other molecules. This proximity enables a synergistic regulation of cellular homeostasis and functions. The formation and maintenance of these contact sites are governed by specific proteins that bring organelle membranes into close apposition, thereby enabling functional crosstalk between cellular compartments. In eukaryotic cells, lipids are primarily synthesized and metabolized within distinct organelles and must be transported through MCS to ensure proper cellular function. Consequently, MCS act as pivotal platforms for lipid synthesis and trafficking, particularly in cancer cells and immune cells within the tumor microenvironment, where dynamic alterations are critical for maintaining lipid homeostasis. This article provides a comprehensive analysis of how these cells exploit membrane contact sites to modulate lipid synthesis, metabolism, and transport, with a specific focus on how MCS-mediated lipid dynamics influence tumor progression. We also examine the differences in MCS and associated molecules across various cancer types, exploring novel therapeutic strategies targeting MCS-related lipid metabolism for the development of anticancer drugs, while also addressing the challenges involved.

Abdominal aortic aneurysm (AAA) is strongly correlated with obesity, partially due to the abnormal expansion of abdominal perivascular adipose tissue (PVAT). Cell death-inducing DNA fragmentation factor-like effector C (CIDEC), also known as fat-specific protein 27 (FSP27) in rodents, is specifically expressed in adipose tissue where it mediates lipid droplet fusion and adipose tissue expansion. Whether and how CIDEC/FSP27 plays a role in AAA pathology remains elusive. Here, we show that FSP27 exacerbates obesity and angiotensin Ⅱ (Ang Ⅱ)-induced AAA progression. FSP27 deficiency in mice inhibited high-fat diet-induced PVAT expansion and inflammation. Both global and adipose tissue-specific FSP27 ablation significantly decreased obesity-related AAA incidence. Deficiency of FSP27 in adipocytes abrogated matrix metalloproteinase-12 (MMP12) expression in aortic tissues. Infiltrated macrophages, which partially colocalize with MMP12, were significantly decreased in the FSP27-deficient aorta. Mechanistically, knockdown of Fsp27 in 3T3-L1 adipocytes inhibited C-C motif chemokine ligand 2 (CCL2) expression and secretion through a c-Jun N-terminal kinase (JNK)-dependent pathway, thereby leading to reduced induction of macrophage migration, while Cidec overexpression rescued this effect. Overall, our study demonstrates that CIDEC/FSP27 in adipose tissue contributes to obesity-related AAA formation, at least in part, by enhancing PVAT inflammation and macrophage infiltration, thus shedding light on its significance as a key regulator in the context of obesity-related AAA.

Hu-lu-su-pian (HLSP) is an oral tablet derived from the active compounds of Cucumis melo L., a traditional Chinese medicine. This contemporary formulation is frequently employed in clinical settings for the management of liver ailments. However, the molecular mechanism by which HLSP affects metabolic dysfunction-associated steatotic liver disease (MASLD) remains unclear. This study aimed to explore the therapeutic potential of HLSP on MASLD and the underlying mechanism.

The researchers used ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) to identify the primary chemical components of HLSP. A mouse model of MASLD induced by AKT was established through hydrodynamic transfection with activated forms of AKT. Serum biochemical indices and liver pathological assessments were employed to evaluate the pharmacodynamic effects of HLSP on MASLD. Transcriptomic analysis of the liver was conducted to detect differentially expressed genes (DEGs). Further examination of significant DEGs and proteins was performed using quantitative real-time polymerase chain reaction (RT-qPCR), Western blotting, and immunohistochemistry (IHC) techniques, respectively. The efficacy and molecular mechanisms of HLSP in MASLD were further explored in HepG2 and Huh-7 cells in the presence of gene overexpression.

From the UPLC-Q-TOF-MS/MS results, we detected fifteen components from HLSP. From the results of serum biochemical indices and hepatic pathology analyses, it is clear that HLSP is effective in treating MASLD. The findings from hepatic transcription studies revealed CIDEA as an essential DEG that facilitates lipid droplet (LD) fusion and enhances de novo fatty acid synthesis from scratch in cases of hepatic steatosis, which HLSP has the potential to counteract. In addition, HLSP significantly reduced lipid accumulation and expression of critical genes for de novo fatty acid synthesis in HepG2 and Huh-7 cells overexpressing CIDEA.

The present study preliminarily suggests that HLSP can ameliorate hepatic steatosis by inhibiting CIDEA-mediated de novo fatty acid synthesis and LD formation, which may offer a potential strategy for treating MASLD.

Actinobacteria belonging to Mycobacterium and Rhodococcus genera are able to synthesize and intracellularly accumulate variable amounts of triacylglycerols (TAG) in the form of lipid droplets (LDs). The lipid storage capacity of LDs in cells is controlled by the balance between lipogenesis and lipolysis. The growth of LDs in bacterial cells may be directly promoted by TAG biosynthesis, whereas TAG degradation might result in the reduction of LD sizes and lipid storage capacity. Therefore, LD formation and turnover have to be precisely regulated to maintain a balanced lipid distribution, coupling gene regulation with the metabolic state of the cell. In eukaryotic cells, LDs have emerged as critical mediators of diverse cellular responses, including fatty acid trafficking and modulation of transcriptional programs. Recent studies performed in mycobacteria and rhodococci suggested the existence of similar crosstalk mechanisms between lipid metabolism, LDs, and gene expression regulation in cells. This review connects and organizes results of different studies in a comprehensive framework for providing evidence of "lipid metabolism-LDs-genomic DNA" crosstalk occurring in TAG-accumulating actinobacteria. We provide examples indicating that bacterial cells evolved sensing mechanisms that detect lipid metabolites changes as indicators of metabolic states, and adapt their transcriptional profiles through epigenetic-like mechanisms mediated by LD-associated proteins. Here, we describe the molecular interconnections of this coupling system and the main role of each component that integrates the information about the cellular metabolic state into the regulation of lipogenesis, LD formation and transcription in oleaginous bacteria.

Lipid droplets (LDs) are dynamic organelles that communicate with other cellular components to orchestrate energetic homeostasis and signal transduction. In skeletal muscle, the presence and importance of LDs have been widely studied in myofibers of both rodents and humans under physiological conditions and in metabolic disorders. However, the role of LDs in myogenic stem cells has only recently begun to be unveiled. In this review we briefly summarize the process of LD biogenesis and degradation in the most prevalent model. We then review recent knowledge on LDs in skeletal muscle and muscle stem cells. We further introduce advanced methodologies for LD imaging and mass spectrometry that have propelled our understanding of the dynamics and heterogeneity of LDs.

High fat diet (HFD) induces the enlargement and accumulation of lipid droplets (LDs) in hepatocytes, thereby influencing the homeostasis of lipid metabolism. Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), a surface protein of LDs, facilitates their fusion and growth, transforming small LDs into larger ones. Lipophagy, a selective form of autophagy, primarily targets small LDs for degradation. Fisetin (FIS), a natural dietary flavonoid present in various fruits and vegetables, has an unclear mechanism for reducing LD accumulation. In this study, we observed that FIS significantly ameliorated HFD-induced lipid accumulation in the hepatocytes of C57BL/6 mice. In further mechanistic studies, we revealed that FFA enhanced the expression of CIDEC, which promoted the fusion of LDs and caused them to become larger. The enlarged LDs could not be degraded by autophagy, which ultimately led to accumulation of LDs. Conversely, FIS alleviated LD accumulation by inhibiting CIDEC-mediated fusion, resulting in smaller LDs that facilitated lipophagy. Additionally, studies indicated that the dysfunction of circadian rhythms is closely related to lipid metabolism. In our study, we showed that HFD and FFA disrupted circadian rhythm in C57BL/6 mouse hepatocytes and AML12 cells, while FIS modified the rhythm disturbances and increased protein expression of the core clocks BMAL1 and CLOCK. We silenced the BMAL1 protein and revealed that si-BMAL1 upregulated CIDEC proteins. These data suggested that FIS might inhibit CIDEC-mediated LD fusion and enhance hepatocyte lipophagy by promoting the expression of rhythm protein BMAL1, thereby alleviating LD accumulation in C57BL/6 and AML12 cells caused by the HFD and FFA. The present study provided novel insights and potential targets for utilizing functional food factors to mitigate the accumulation of LD in hepatocytes.

This study investigates the role of actin polymerization and Myh9 in mediating lipid droplet (LD) fission during rosiglitazone-induced browning of white adipocytes. The aim is to understand how LD splitting might contribute to the beige conversion of white adipose tissue, providing insights into adipocyte plasticity and metabolic regulation.

C3H10 T1/2-differentiated adipocytes were used as a classical model to study white adipocyte browning. Rosiglitazone was applied to induce browning, and the interactions between LDs and actin, as well as the distribution of Myh9, were assessed using immunofluorescence and Western blotting. In vivo, we employed a microfilament inhibitor to block actin polymerization in cold-stimulated mice and evaluated changes in LD morphology and browning. Furthermore, dynamic live-cell imaging using confocal microscopy was conducted to observe the real-time behavior of LDs during the browning process and to determine whether they undergo fission.

Our results demonstrate that rosiglitazone significantly induces LD size reduction, a process correlated with the increased contact of LDs with microfilaments. Inhibition of actin polymerization prevented both the reduction in LD size and the browning of white adipocytes, indicating that actin plays a critical role. Myh9 was enriched at the LD fission sites, forming a structure resembling a contractile ring. Overexpression of Myh9 promoted the shrinkage of LD, suggesting that it may be involved in LD fission.

This study identifies actin and Myh9 as key regulators of LD fission in rosiglitazone-induced browning of white adipocytes, offering new insights into the cellular mechanisms of adipocyte plasticity. The findings propose a novel pathway by which LD dynamics contribute to the beige conversion of white fat, with potential implications for metabolic disease therapies targeting adipocyte function and energy expenditure.

Complex genetic and dietary cues contribute to the development of obesity, but how these are integrated on a molecular level is incompletely understood. Here, we show that PPARγ supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, a membrane-bound O-acyltransferase that enriches diet-derived omega-6 ( n -6) polyunsaturated fatty acids (PUFAs) in the phospholipidome. In high-fat diet-fed mice, lowering membrane n -6 PUFA levels by adipocyte-specific Lpcat3 knockout ( Lpcat3 AKO ) or by dietary lipid manipulation leads to dysfunctional triglyceride (TG) storage, ectopic fat deposition and insulin resistance. Aberrant lipolysis of stored TGs in Lpcat3 AKO adipose tissues instigates a non-canonical adaptive response that engages a futile lipid cycle to increase energy expenditure and limit further body weight gain. Mechanistically, we find that adipocyte LPCAT3 activity promotes TG storage by selectively enriching n -6 arachidonoyl-phosphatidylethanolamine at the ER-lipid droplet interface, which in turn favours the budding of large droplets that exhibit greater resistance to ATGL-dependent hydrolysis. Thus, our study highlights the PPARγ-LPCAT3 pathway as a molecular link between dietary n -6 PUFA intake, adipose expandability and systemic energy balance.

Nonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin-degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo-endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR.

We investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress-induced UPR and lipid accumulation in the liver.

We found that IDE-deficient mice display higher hepatic triglyceride content along with higher inositol-requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress-induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis.

These results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.

To investigate the effects of β-hydroxybutyrate (BHB) and melatonin on brown adipose tissue (BAT) plasticity in rats fed a high-fat diet (HFD).

We employed a 7-week experimental design for a study on 30 male Sprague-Dawley rats divided into five groups: (1) a control-diet fed group; (2) a high-fat diet (HFD)-fed group; (3) a group that received an HFD and a BHB solution in their drinking water; (4) a group that received an HFD with 10 mg/kg/day melatonin in their drinking water; and (5) a group that received an HFD and were also treated with the combination of BHB and melatonin. Following the treatment period, biochemical indices, gene expression levels of key thermogenic markers (including uncoupling protein 1 [UCP1], PR domain containing 16 [PRDM16], Cidea, fat-specific protein 27 [Fsp27], and metallothionein 1 [MT1]), and stereological assessments of BAT were evaluated.

Treatment with BHB and melatonin significantly boosted blood ketone levels, improved lipid profiles, and reduced weight gain from an HFD. It also downregulated genes linked to WAT, namely, Cidea and Fsp27, and upregulated key BAT markers, including UCP1, PRDM16 and peroxisome proliferator-activated receptor-gamma coactivator-1-alpha. Additionally, the co-treatment increased MT1 receptor expression and enhanced the structural density of BAT.

The combined oral administration of BHB and melatonin successfully prevented the whitening of BAT in obese rats fed an HFD, indicating its potential as a therapeutic strategy for obesity-related BAT dysfunction. The synergistic effects of this treatment underscore the potential of a combined approach to address BAT dysfunction in obesity.

Lipid droplets (LDs) are dynamic storage organelles with central roles in lipid and energy metabolism. They consist of a core of neutral lipids, such as triacylglycerol, which is surrounded by a monolayer of phospholipids and specialized surface proteins. The surface composition determines many of the LD properties, such as size, subcellular distribution, and interaction with partner organelles. Considering the diverse energetic and metabolic demands of various cell types, it is not surprising that LDs are highly heterogeneous within and between cell types. Despite their diversity, all LDs share a common biogenesis mechanism. However, adipocytes have evolved specific adaptations of these basic mechanisms, enabling the regulation of lipid and energy metabolism at both the cellular and organismal levels. Here, we discuss recent advances in the understanding of both the general mechanisms of LD biogenesis and the adipocyte-specific adaptations controlling these fascinating organelles.

Lipid droplet (LD) is a metabolically active organelle, which changes dynamically with the metabolic state and energy requirements of cells. Proteins that either insert into the LD phospholipid monolayer or are present in the cytoplasm, playing a crucial role in lipid homeostasis and signaling regulation, are known as LD-associated proteins.

The keywords "lipid droplets" and "metabolic diseases" were used to obtain literature on LD metabolism and pathological mechanism. After searching databases including Scopus, OVID, Web of Science, and PubMed from 2013 to 2024 using terms like "lipid droplets", "lipid droplet-associated proteins", "fatty liver disease", "diabetes", "diabetic kidney disease", "obesity", "atherosclerosis", "hyperlipidemia", "natural drug monomers" and "natural compounds", the most common natural compounds were identified in about 954 articles. Eventually, a total of 91 studies of 10 natural compounds reporting in vitro or in vivo studies were refined and summarized.

The most frequently used natural compounds include Berberine, Mangostin, Capsaicin, Caffeine, Genistein, Epigallocatechin-3-gallate, Chlorogenic acid, Betaine, Ginsenoside, Resveratrol. These natural compounds interact with LD-associated proteins and help ameliorate abnormal LDs in various metabolic diseases.

Natural compounds involved in the regulation of LDs and LD-associated proteins hold promise for treating metabolic diseases. Further research into these interactions may lead to new therapeutic applications.

Lipid droplets (LDs), which are active organelles, derive from the monolayer membrane of the endoplasmic reticulum and encapsulate neutral lipids internally. LD-associated proteins like RAB, those in the PLIN family, and those in the CIDE family participate in LD formation and development, and they are active players in various diseases, organelles, and metabolic processes (i.e., obesity, non-alcoholic fatty liver disease, and autophagy). Our synthesis on existing research includes insights from the formation of LDs to their mechanisms of action, to provide an overview needed for advancing research into metabolic diseases and lipid metabolism.

Hepatic steatosis is a common finding in liver histopathology and the hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), whose global prevalence is rising.

To review the histopathology of hepatic steatosis and its mechanisms of development and to identify common and rare disease associations.

We reviewed literature on the basic science of lipid droplet (LD) biology and clinical research on acute and chronic liver diseases associated with hepatic steatosis using the PubMed database.

A variety of genetic and environmental factors contribute to the development of chronic hepatic steatosis or steatotic liver disease, which typically appears macrovesicular. Microvesicular steatosis is associated with acute mitochondrial dysfunction and liver failure. Fat metabolic processes in hepatocytes whose dysregulation leads to the development of steatosis include secretion of lipoprotein particles, uptake of remnant lipoprotein particles or free fatty acids from blood, de novo lipogenesis, oxidation of fatty acids, lipolysis and lipophagy. Hepatic insulin resistance is a key feature of MASLD. Seipin is a polyfunctional protein that facilitates LD biogenesis. Assembly of hepatitis C virus takes place on LD surfaces. LDs make important, functional contact with the endoplasmic reticulum and other organelles.

Diverse liver pathologies are associated with hepatic steatosis, with MASLD being the most important contributor. The biogenesis and dynamics of LDs in hepatocytes are complex and warrant further investigation. Organellar interfaces permit co-regulation of lipid metabolism to match generation of potentially toxic lipid species with their LD depot storage.

Lipid droplets (LDs) are dynamic organelles that store neutral lipids and are closely linked to obesity. Previous studies have suggested that Lycium barbarum polysaccharide (LBP) supplements can ameliorate obesity, but the underlying mechanisms remain unclear. In this study, we hypothesize that LBP alleviates LD accumulation in adipose tissue (AT) by inhibiting fat-specific protein 27 (Fsp27) through an activating transcription factor-6 (ATF6)/small-molecule sirtuin 1 (SIRT1)-dependent mechanism. LD accumulation in AT is induced in high-fat diet (HFD)-fed mice, and differentiation of 3T3-L1 preadipocytes (PAs) is induced. The ability of LBP to alleviate LD accumulation and the possible underlying mechanism are then investigated both in vivo and in vitro. The influences of LBP on the expressions of LD-associated genes ( ATF6 and Fsp27) are also detected. The results show that HFD and PA differentiation markedly increase LD accumulation in ATs and adipocytes, respectively, and these effects are markedly suppressed by LBP supplementation. Furthermore, LBP significantly activates SIRT1 and decreases ATF6 and Fsp27 expressions. Interestingly, the inhibitory effects of LBP are either abolished or exacerbated when ATF6 is overexpressed or silenced, respectively. Furthermore, SIRT1 level is transcriptionally regulated by LBP through opposite actions mediated by ATF6. Collectively, our findings suggest that LBP supplementation alleviates obesity by ameliorating LD accumulation, which might be partially mediated by an ATF6/SIRT1-dependent mechanism.

Several tissues contribute to the onset and advancement of knee osteoarthritis (OA). One tissue type that is worthy of closer evaluation, particularly in the context of sex, is the infrapatellar fat pad (IFP). We previously demonstrated that removal of the IFP had short-term beneficial effects for a cohort of male Dunkin-Hartley guinea pigs. The present project was designed to elucidate the influence of IFP removal in females of this OA-prone strain. It was hypothesized that resection of the IFP would reduce the development of OA in knees of a rodent model predisposed to the disease.

Female guinea pigs (n=16) were acquired at an age of 2.5 months. Surgical removal of the IFP and associated synovium complex (IFP/SC) was executed at 3 months of age. One knee had the IFP/SC resected; a comparable sham surgery was performed on the contralateral knee. All animals were subjected to voluntary enclosure monitoring and dynamic weight-bearing, as well as compulsory treadmill-based gait analysis monthly; baseline data was collected prior to surgery. Guinea pigs were euthanized at 7 months. Knees from eight animals were evaluated via histology, mRNA expression, and immunohistochemistry (IHC); knees from the remaining eight animals were allocated to microcomputed tomography (microCT), biomechanical analyses (whole joint testing and indentation relaxation testing), and atomic absorption spectroscopy (AAS).

Fibrous connective tissue (FCT) replaced the IFP/SC. Mobility/gait data indicated that unilateral IFP/SC removal did not affect bilateral hindlimb movement. MicroCT demonstrated that osteophytes were not a significant feature of OA in this sex; however, trabecular thickness (TbTh) in medial femorae decreased in knees containing the FCT. Histopathology scores were predominantly influenced by changes in the lateral tibia, which demonstrated that histologic signs of OA were increased in knees containing the native IFP/SC versus those with the FCT. Similarly, indentation testing demonstrated higher instantaneous and equilibrium moduli in the lateral tibial articular cartilage of control knees with native IFPs. AAS of multiple tissue types associated with the knee revealed that zinc was the major trace element influenced by removal of the IFP/SC.

Our data suggest that the IFP/SC is a significant component driving knee OA in female guinea pigs and that resection of this tissue prior to disease has short-term benefits. Specifically, the formation of the FCT in place of the native tissue resulted in decreased cartilage-related OA changes, as demonstrated by reduced Osteoarthritis Research Society International (OARSI) histology scores, as well as changes in transcript, protein, and cartilage indentation analyses. Importantly, this model provides evidence that sex needs to be considered when investigating responses and associated mechanisms seen with this intervention.

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.

Lipid metabolism is important for the maintenance of physiological homeostasis. Several members of the small ubiquitin-like modifier (SUMO)-specific protease (SENP) family have been reported as the regulators of lipid homeostasis. However, the function of Senp7 in lipid metabolism remains unclear. In this study, we generated both conventional and adipocyte-specific Senp7 KO mice to characterize the role of Senp7 in lipid metabolism homeostasis. Both Senp7-deficient mice displayed reduced white adipose tissue mass and decreased size of adipocytes. By analyzing the lipid droplet morphology, we demonstrated that the lipid droplet size was significantly smaller in Senp7-deficient adipocytes. Mechanistically, Senp7 could deSUMOylate the perilipin family protein Plin4 to promote the lipid droplet localization of Plin4. Our results reveal an important role of Senp7 in the maturation of lipid droplets via Plin4 deSUMOylation.

The cell death-inducing DFF45-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec/Fsp27, regulate various aspects of lipid homeostasis, including lipid storage, lipolysis, and lipid secretion. This review focuses on the physiological roles of CIDE proteins based on studies on knockout mouse models and human patients bearing CIDE mutations. The primary cellular function of CIDE proteins is to localize to lipid droplets (LDs) and to control LD fusion and growth across different cell types. We propose a four-step process of LD fusion, characterized by (a) the recruitment of CIDE proteins to the LD surface and CIDE movement, (b) the enrichment and condensate formation of CIDE proteins to form LD fusion plates at LD-LD contact sites, (c) lipid transfer through lipid-permeable passageways within the fusion plates, and (d) the completion of LD fusion. Lastly, we outline CIDE-interacting proteins as regulatory factors, as well as their contribution in LD fusion.

Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near-atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD-related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology.

Many metabolic diseases are caused by disorders of lipid homeostasis. CIDEC, a lipid droplet (LD)-associated protein, plays a critical role in controlling LD fusion and lipid storage. However, regulators of CIDEC remain largely unknown. Here, we established a homogeneous time-resolved fluorescence (HTRF)-based high-throughput screening method and identified LPXN as a positive regulatory candidate for CIDEC. LPXN and Hic-5, the members of the Paxillin family, are focal adhesion adaptor proteins that contribute to the recruitment of specific kinases and phosphatases, cofactors, and structural proteins, participating in the transduction of extracellular signals into intracellular responses. Our data showed that Hic-5 and LPXN significantly increased the protein level of CIDEC and enhanced CIDEC stability not through triacylglycerol synthesis and FAK signaling pathways. Hic-5 and LPXN reduced the ubiquitination of CIDEC and inhibited its proteasome degradation pathway. Furthermore, Hic-5 and LPXN enlarged LDs and promoted lipid storage in adipocytes. Therefore, we identified Hic-5 and LPXN as novel regulators of CIDEC. Our current findings also suggest intervention with Hic-5 and LPXN might ameliorate ectopic fat storage by enhancing the lipid storage capacity of white adipose tissues.

Lipid droplets (LDs) are intracellular organelles that play important roles in cellular lipid metabolism; they change their sizes and numbers in response to both intracellular and extracellular signals. Changes in LD size reflect lipid synthesis and degradation and affect many cellular activities, including energy supply and membrane synthesis. Here, we focused on the function of the endoplasmic reticulum-plasma membrane tethering protein Ice2 in LD dynamics in the fungal pathogen Candida albicans (C. albicans). Nile red staining and size quantification showed that the LD size increased in the ice2Δ/Δ mutant, indicating the critical role of Ice2 in the regulation of LD dynamics. A lipid content analysis further demonstrated that the mutant had lower phosphatidylcholine levels. As revealed with GFP labeling and fluorescence microscopy, the methyltransferase Cho2, which is involved in phosphatidylcholine synthesis, had poorer localization in the plasma membrane in the mutant than in the wild-type strain. Interestingly, the addition of the phosphatidylcholine precursor choline led to the recovery of normal-sized LDs in the mutant. These results indicated that Ice2 regulates LD size by controlling intracellular phosphatidylcholine levels and that endoplasmic reticulum-plasma membrane tethering proteins play a role in lipid metabolism regulation in C. albicans. This study provides significant findings for further investigation of the lipid metabolism in fungi.

Interorganelle contacts facilitate material exchanges and sustain the structural and functional integrity of organelles. Lipid droplets (LDs) of adipocytes are responsible for energy storage and mobilization responding to body needs. LD biogenesis defects compromise the lipid-storing capacity of adipocytes, resulting in ectopic lipid deposition and metabolic disorders, yet how the uniquely large LDs in adipocytes attain structural and functional maturation is incompletely understood. Here we show that the mammalian adipocyte-specific protein CLSTN3B is crucial for adipocyte LD maturation. CLSTN3B employs an arginine-rich segment to promote extensive contact and hemifusion-like structure formation between the endoplasmic reticulum (ER) and LD, allowing ER-to-LD phospholipid diffusion during LD expansion. CLSTN3B ablation results in reduced LD surface phospholipid density, increased turnover of LD-surface proteins, and impaired LD functions. Our results establish the central role of CLSTN3B in the adipocyte-specific LD maturation pathway that enhances lipid storage and maintenance of metabolic health under caloric overload.

Choline deficiency causes disorders including hepatic abnormalities and is associated with an increased risk of multiple types of cancer. Here, by choline-free diet-associated RNA-Seq analyses, we found that the tumor suppressor p53 drives the Kennedy pathway via PCYT1B to control the growth of lipid droplets (LDs) and their fueling role in tumorigenesis. Mechanistically, through upregulation of PCYT1B, p53 channeled depleted choline stores to phosphatidylcholine (PC) biosynthesis during choline starvation, thus preventing LD coalescence. Cells lacking p53 failed to complete this response to choline depletion, leading to hepatic steatosis and tumorigenesis, and these effects could be reversed by enforcement of PCYT1B expression or restoration of PC abundance. Furthermore, loss of p53 or defects in the Kennedy pathway increased surface localization of hormone-sensitive lipase on LDs to release specific fatty acids that fueled tumor cells in vivo and in vitro. Thus, p53 loss leads to dysregulation of choline metabolism and LD growth and couples perturbed LD homeostasis to tumorigenesis.

The ratio of free fatty acid (FFA) turnover decreases significantly with the expansion of white adipose tissue. Adipose tissue and dietary saturated fatty acid levels significantly correlate with an increase in fat cell size and number. The G0/G1 switch gene 2 increases lipid content in adipocytes and promotes adipocyte hypertrophy through the restriction of triglyceride (triacylglycerol: TAG) turnover. Hypoxia in obese adipose tissue due to hypertrophic adipocytes results in excess deposition of extracellular matrix (ECM) components. Cluster of differentiation (CD) 44, as the main receptor of the extracellular matrix component regulates cell-cell and cell-matrix interactions including diet-induced insulin resistance. Excess TAGs, sterols, and sterol esters are surrounded by the phospholipid monolayer surface and form lipid droplets (LDs). Once LDs are formed, they grow up because of the excessive amount of intracellular FFA stored and reach a final size. The ratio of FFA turnover/lipolysis decreases significantly with increases in the degree of obesity. Dysfunctional adipose tissue is unable to expand further to store excess dietary lipids, increased fluxes of plasma FFAs lead to ectopic fatty acid deposition and lipotoxicity. Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Obesity-associated adipocyte death exhibits feature of necrosis-like programmed cell death. Adipocyte death is a prerequisite for the transition from hypertrophic to hyperplastic obesity. Increased adipocyte number in obesity has life-long effects on white adipose tissue mass. The positive correlation between the adipose tissue volume and magnetic resonance imaging proton density fat fraction estimation is used for characterization of the obesity phenotype, as well as the risk stratification and selection of appropriate treatment strategies. In obese patients with type 2 diabetes, visceral adipocytes exposed to chronic/intermittent hyperglycemia develop a new microRNAs' (miRNAs') expression pattern. Visceral preadipocytes memorize the effect of hyperglycemia via changes in miRNAs' expression profile and contribute to the progression of diabetic phenotype. Nonsteroidal anti-inflammatory drugs, metformin, and statins can be beneficial in treating the local or systemic consequences of white adipose tissue inflammation. Rapamycin inhibits leptin-induced LD formation. Collectively, in this chapter, the concept of adipose tissue remodeling in response to adipocyte death or adipogenesis, and the complexity of LD interactions with the other cellular organelles are reviewed. Furthermore, clinical perspective of fat cell turnover in obesity is also debated.

Lipid droplets (LDs) are dynamic lipid storage organelles that can sense and respond to changes in systemic energy balance. The size and number of LDs are controlled by complex and delicate mechanisms, among which, whether and which SNARE proteins mediate LD fusion, and the mechanisms governing this process remain poorly understood. Here we identified a SNARE complex, syntaxin 18 (STX18)-SNAP23-SEC22B, that is recruited to LDs to mediate LD fusion. STX18 targets LDs with its transmembrane domain spanning the phospholipid monolayer twice. STX18-SNAP23-SEC22B complex drives LD fusion in adiposome lipid mixing and content mixing in vitro assays. CIDEC/FSP27 directly binds STX18, SEC22B, and SNAP23, and promotes the lipid mixing of SNAREs-reconstituted adiposomes by promoting LD clustering. Knockdown of STX18 in mouse liver via AAV resulted in smaller liver and reduced LD size under high-fat diet conditions. All these results demonstrate a critical role of the SNARE complex STX18-SNAP23-SEC22B in LD fusion.

Lipid droplets are not only lipid storage sites but also are closely related to lipid metabolism. Lipid droplet growth increases lipid storage capacity and suppresses lipolysis via lipase associated with the lipid droplet surface. The cell death-inducing DFF45-like effector (CIDE) family of proteins mediates lipid droplet fusion, which mainly contributes to lipid droplet growth. We previously demonstrated small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) plays important roles in lipid metabolism and induction/maintenance of adipogenesis. In this study, we determined whether SENP2 regulates lipid droplet size in adipocytes. Overexpression of SENP2 increased lipid droplet size in differentiated 3T3-L1 adipocytes and facilitated CIDEA transcription. We found SENP2 increased CIDEA expression mainly through desumoylation of estrogen-related receptor α (ERRα), which acted in coordination with peroxisome proliferator-activated receptor γ-coactivator α. In addition, palmitate treatment increased SENP2 and CIDEA mRNA levels. Specific small interfering RNA-mediated knockdown of SENP2, as well as ERRα knockdown, eliminated palmitate-induced CIDEA expression. These results suggest SENP2 enhances CIDEA expression by modulating ERRα when SENP2 is upregulated, such as after palmitate treatment, to increase lipid droplet size in adipocytes.

Metabolic reprogramming has been considered a core hallmark of cancer, in which excessive accumulation of lipids promote cancer initiation, progression and metastasis. Lipid metabolism often includes the digestion and absorption of dietary fat, and the ways in which cancer cells utilize lipids are often influenced by the complex interactions within the tumor microenvironment. Among multiple cancer risk factors, obesity has a positive association with multiple cancer types, while diets like calorie restriction and fasting improve health and delay cancer. Impact of these diets on tumorigenesis or cancer prevention are generally studied on cancer cells, despite heterogeneity of the tumor microenvironment. Cancer cells regularly interact with these heterogeneous microenvironmental components, including immune and stromal cells, to promote cancer progression and metastasis, and there is an intricate metabolic crosstalk between these compartments. Here, we focus on discussing fat metabolism and response to dietary fat in the tumor microenvironment, focusing on both immune and stromal components and shedding light on therapeutic strategies surrounding lipid metabolic and signaling pathways.

Tissues within an organism and even cell types within a tissue can age with different velocities. However, it is unclear whether cells of one type experience different aging trajectories within a tissue depending on their spatial location. Here, we used spatial transcriptomics in combination with single-cell ATAC-seq and RNA-seq, lipidomics and functional assays to address how cells in the male murine liver are affected by age-related changes in the microenvironment. Integration of the datasets revealed zonation-specific and age-related changes in metabolic states, the epigenome and transcriptome. The epigenome changed in a zonation-dependent manner and functionally, periportal hepatocytes were characterized by decreased mitochondrial fitness, whereas pericentral hepatocytes accumulated large lipid droplets. Together, we provide evidence that changing microenvironments within a tissue exert strong influences on their resident cells that can shape epigenetic, metabolic and phenotypic outputs.

While disorders in lipid metabolism have been associated with aging and age-related diseases, how lipid metabolism is regulated during aging is poorly understood. Here, we characterize the Drosophila endoribonuclease CG2145, an ortholog of mammalian EndoU that we named Age-related lipid regulator (Arlr), as a regulator of lipid homeostasis during aging. In adult adipose tissues, Arlr is necessary for maintenance of lipid storage in lipid droplets (LDs) as flies age, a phenotype that can be rescued by either high-fat or high-glucose diet. Interestingly, RNA-seq of arlr mutant adipose tissues and RIP-seq suggest that Arlr affects lipid metabolism through the degradation of the mRNAs of lipolysis genes - a model further supported by the observation that knockdown of Lsd-1, regucalcin, yip2 or CG5162, which encode genes involved in lipolysis, rescue the LD defects of arlr mutants. In addition, we characterize DendoU as a functional paralog of Arlr and show that human ENDOU can rescue arlr mutants. Altogether, our study reveals a role of ENDOU-like endonucleases as negative regulator of lipolysis.

Membrane contact sites (MCSs) are areas of close proximity between organelles, implicated in transport of small molecules and in organelle biogenesis. Lipid transfer proteins at MCSs facilitate the distribution of lipid species between organelle membranes. Such exchange processes rely on the apposition of two different membranes delimiting distinct compartments and a cytosolic intermembrane space. Maintaining organelle identity while transferring molecules therefore implies control over MCS architecture both on the ultrastructural and molecular levels. Factors including intermembrane distance, density of resident proteins, and contact surface area fine-tune MCS function. Furthermore, the structural arrangement of lipid transfer proteins and associated proteins underpins the molecular mechanisms of lipid fluxes at MCSs. Thus, the architecture of MCSs emerges as an essential aspect of their function.

Once regarded as inert organelles with limited and ill-defined roles, lipid droplets (LDs) have emerged as dynamic entities with multifaceted functions within the cell. Recent research has illuminated their pivotal role as primary energy reservoirs in the form of lipids, capable of being metabolized to meet cellular energy demands. Their high dynamism is underscored by their ability to interact with numerous cellular organelles, notably the endoplasmic reticulum (the site of LD genesis) and mitochondria, which utilize small LDs for energy production. Beyond their contribution to cellular bioenergetics, LDs have been associated with viral infections. Evidence suggests that viruses can co-opt LDs to facilitate their infection cycle. Furthermore, recent discoveries highlight the role of LDs in modulating the host's immune response. Observations of altered LD levels during viral infections suggest their involvement in disease pathophysiology, potentially through production of proinflammatory mediators using LD lipids as precursors. This review explores these intriguing aspects of LDs, shedding light on their multifaceted nature and implications in viral interactions and disease development.

Mammalian lipid droplets (LDs) are specialized cytosolic organelles consisting of a neutral lipid core surrounded by a membrane made up of a phospholipid monolayer and a specific population of proteins that varies according to the location and function of each LD. Over the past decade, there have been significant advances in the understanding of LD biogenesis and functions. LDs are now recognized as dynamic organelles that participate in many aspects of cellular homeostasis plus other vital functions. LD biogenesis is a complex, highly-regulated process with assembly occurring on the endoplasmic reticulum although aspects of the underpinning molecular mechanisms remain elusive. For example, it is unclear how many enzymes participate in the biosynthesis of the neutral lipid components of LDs and how this process is coordinated in response to different metabolic cues to promote or suppress LD formation and turnover. In addition to enzymes involved in the biosynthesis of neutral lipids, various scaffolding proteins play roles in coordinating LD formation. Despite their lack of ultrastructural diversity, LDs in different mammalian cell types are involved in a wide range of biological functions. These include roles in membrane homeostasis, regulation of hypoxia, neoplastic inflammatory responses, cellular oxidative status, lipid peroxidation, and protection against potentially toxic intracellular fatty acids and lipophilic xenobiotics. Herein, the roles of mammalian LDs and their associated proteins are reviewed with a particular focus on their roles in pathological, immunological and anti-toxicological processes.