ApoL6 associates with lipid droplets and disrupts Perilipin1-HSL interaction to inhibit lipolysis

White adipose tissue (WAT) stores excess energy as triacylglycerol (TAG) in lipid droplets (LD) and mobilizes fatty acids during energy deprivation via lipolysis. Lipolysis proceeds through a regulated cascade catalyzed by ATGL, HSL, and MGL, and is controlled by hormonal signals (catecholamines, insulin) that modulate PKA activity and phosphorylation of lipases and LD-associated proteins. Perilipin 1 (Plin1) is a key LD scaffold that, upon phosphorylation, coordinates assembly of the lipolytic machinery including CGI-58 and ATGL, and recruits phosphorylated HSL to LD. Dysregulation of adipose lipolysis contributes to obesity, insulin resistance, and ectopic lipid deposition. The study aims to identify and characterize LD-associated proteins that regulate adipocyte TAG metabolism, focusing on ApoL6, and to determine its mechanism of action and physiological impact on adiposity and metabolic health.

Prior work has established the enzymatic steps of lipolysis (ATGL for TAG to DAG, HSL for DAG to MAG, MGL for MAG to FA) and the importance of PKA-mediated phosphorylation of HSL, Plin1, ATGL (via AMPK) and CGI-58 in regulating lipase localization and activity. Plin1 functions as a gatekeeper, suppressing basal lipolysis and enabling stimulated lipolysis via phosphorylation-dependent recruitment of HSL. G0/G1 switch gene 2 (GOS2) is an endogenous ATGL inhibitor. Genetic and pharmacological studies in rodents and humans implicate lipolytic components as therapeutic targets. The apolipoprotein L family shares amphipathic helices and membrane/coiled-coil domains; certain members have intracellular roles in lipid handling. GWAS have linked SNPs near ApoL6 to triglyceride and HDL levels, suggesting a potential role in lipid metabolism.

• Gene and protein expression: RT-qPCR, Northern blotting, and immunoblotting were used to assess ApoL6 expression across tissues (iWAT, eWAT, BAT) and cellular fractions (adipocytes vs stromal vascular fraction), during 3T3-L1 adipocyte differentiation, and under fasting/refeeding. Promoter analysis identified E-box and SRE motifs; a −1 kb ApoL6 promoter luciferase reporter was co-transfected with USF-1 or SREBP1c to assess transcriptional activation.
• Subcellular localization: ApoL6-GFP was expressed in differentiated 3T3-L1 adipocytes; confocal microscopy with LipidTOX staining and sucrose gradient fractionation identified LD association.
• Protein-lipid interactions: Protein-lipid overlay assays (membrane strips) tested binding of ApoL6-GST and mutants to phospholipids (PI(4,5)P2, PI(3,4,5)P3, PI(4)P, PA, etc.). Mutational analysis of a cationic motif in the ApoL6 C-terminus defined determinants of PIP2 binding.
• Gain- and loss-of-function in adipocytes: Adenoviral and lentiviral overexpression of ApoL6 (mouse and human) in 3T3-L1 and human fibroblast–derived adipocytes; shRNA knockdown of ApoL6 in both models. Outcomes included LD size (LipidTOX imaging), TAG content (biochemical assays), and pulse-chase with [U-14C] palmitate followed by TLC to quantify labeled TAG/DAG/MAG/FFA.
• Lipolysis assays: Measurement of FFA and glycerol release in basal and isoproterenol-stimulated conditions; pharmacological inhibition using Atglistatin (ATGL inhibitor) and CAY10499 (HSL inhibitor) to test dependency.
• Mouse models: Global ApoL6 knockout (CRISPR-Cas9, 11 bp deletion in exon 3) and adipocyte-specific ApoL6 overexpression (aP2-ApoL6 TG; adipoQ-ApoL6 TG). Phenotyping included body weight, EchoMRI (fat/lean mass), tissue weights, WAT histology (H&E), adipocyte size quantification, serum metabolites (FFA, TAG, cholesterol, HDL/LDL), glucose tolerance (GTT) and insulin tolerance (ITT), and WAT inflammation markers. Food intake monitored. Both sexes examined in selected experiments.
• In vivo lipolysis: Isoproterenol (10 mg/kg) challenge with serial blood sampling for glycerol to assess whole-body lipolysis.
• Ex vivo primary adipocytes: Dispersed adipocytes from WAT were used for lipolysis assays ± isoproterenol and inhibitors.
• Protein-protein interactions: Co-immunoprecipitation (Co-IP) from HEK293/293FT, 3T3-L1, and mouse WAT; tandem IP of LD-associated protein fractions; native PAGE to detect high molecular weight complexes; mass spectrometry identification of complex components.
• Direct binding: GST pull-down using ApoL6-GST and in vitro translated 35S-labeled Plin1, HSL, ATGL; pull-down with purified proteins. Bimolecular fluorescence complementation (BiFC) using Venus fragments fused to ApoL6 and Plin1 to visualize interaction on LD.
• Domain mapping: ApoL6 deletion constructs mapped Plin1-interacting region (C-terminal aa 211–321). Plin1 deletion constructs (N-terminal aa 1–280, C-terminal aa 281–517) identified the ApoL6-binding region. Tested Plin1 phosphomimetic (S81D/S222D/S276D) for effects on interactions.
• Signaling assays: Immunoblotting for ATGL S406 phosphorylation in WAT of ApoL6 transgenic mice.
• Statistics: Data reported as mean ± SD. Student’s t test, multiple t tests, and two-way ANOVA were used depending on design (e.g., lipolysis, GTT/ITT, body weight). P < 0.05 considered significant. Experiments typically repeated at least twice with stated n per figure.

• ApoL6 is adipose-enriched and nutritionally regulated: ApoL6 mRNA/protein is primarily in WAT adipocytes (low in SVF), modest in BAT, induced upon feeding and suppressed by fasting; elevated in ob/ob mice. Promoter assays show activation by USF-1 and SREBP1c.
• LD localization via phosphoinositide binding: ApoL6 localizes to LD in adipocytes; subcellular fractionation confirms LD enrichment. ApoL6-GST binds selectively to PI(4)P, PI(4,5)P2, and PI(3,4,5)P3, with preference for PI(4,5)P2; binding requires a cationic motif in the C-terminus (aa 288–292). A charge-reversal mutant loses PIP2 binding and instead binds PA. ApoL6-PIP2 binding is not required for its anti-lipolytic function.
• ApoL6 promotes TAG/LD accumulation without affecting adipogenesis/lipogenesis: Overexpression in 3T3-L1 adipocytes enlarges LD and increases TAG; [14C]-TAG increases by ~60%, with lower labeled FFA and no accumulation of DAG/MAG. Human adipocytes overexpressing ApoL6 show 2.2-fold higher TAG. Knockdown reduces LD size and decreases TAG by ~25% in 3T3-L1 and reduces TAG in human adipocytes.
• ApoL6 inhibits lipolysis: In 3T3-L1 adipocytes, ApoL6 overexpression reduces FFA release by ~30% in basal and stimulated conditions; glycerol release decreases by ~30% (basal) and ~60% (stimulated). Human adipocytes show significantly reduced stimulated FFA release with ApoL6 overexpression. With ATGL or HSL inhibition, differences between control and ApoL6 OE disappear, indicating dependence on canonical lipases. ApoL6 knockdown increases lipolysis in both basal and stimulated states in mouse and human adipocytes; ApoL6 KO MEF-derived adipocytes show higher FFA release; adenoviral ApoL6 rescues phenotype.
• In vivo loss-of-function protects against obesity and improves metabolism: ApoL6 KO mice (both sexes) on HFD gain less weight, with lower fat mass and smaller adipocytes; no change in lean mass or food intake. KO mice show improved GTT and ITT after HFD, lower fed serum TAG and total cholesterol, similar fed FFA but higher fasted FFA. Dispersed adipocytes from KO WAT have higher basal and stimulated lipolysis; pharmacologic inhibitors blunt lipolysis similarly in WT and KO.
• In vivo gain-of-function increases adiposity and induces insulin resistance: aP2-ApoL6 and adipoQ-ApoL6 transgenic mice overexpress ApoL6 specifically in adipose tissue. On chow, aP2-TG show higher body and fat mass with larger adipocytes. On HFD, both TG lines have higher BW and fat mass, increased WAT depot weights, larger adipocytes, elevated serum TAG and cholesterol/LDL, increased WAT inflammatory markers, impaired GTT and ITT, and hepatic steatosis (Oil Red O, TAG). Isoproterenol-stimulated in vivo lipolysis is blunted in aP2-TG (WT ~16-fold glycerol rise at 15 min vs TG ~4-fold).
• Mechanism: ApoL6 forms a high-MW lipolytic complex with Plin1 and HSL on LD. Co-IP shows ApoL6 associates with Plin1, HSL, and ATGL, but GST pull-down demonstrates direct binding only to Plin1. BiFC localizes ApoL6–Plin1 interaction to LD. Domain mapping shows ApoL6 C-terminus (aa 211–321) binds the N-terminal domain of Plin1 (aa 1–280). ApoL6 overexpression disrupts Plin1–HSL interaction in cells and adipocytes; C-terminally truncated ApoL6 (N-ApoL6) fails to disrupt. The ApoL6–Plin1 interaction and lipolysis inhibition are independent of Plin1 phosphorylation-mimic status. Plin1 knockdown abolishes ApoL6’s inhibitory effect on lipolysis. Despite HSL targeting, no DAG accumulation is observed; ATGL S406 phosphorylation is markedly reduced in ApoL6-TG WAT during fasting, suggesting broader suppression of lipolysis.
• Model: In fed state, ApoL6 is induced and binds Plin1 N-terminus, preventing HSL docking and suppressing lipolysis; during fasting, ApoL6 decreases, permitting Plin1–HSL interaction and lipolysis activation.

The study identifies ApoL6 as a previously unrecognized, adipose-enriched LD-associated regulator of lipolysis that is sensitive to nutritional status. Mechanistically, ApoL6’s C-terminal domain directly binds the N-terminus of Plin1, preventing the critical Plin1–HSL interaction required for stimulated lipolysis. This explains reduced FFA/glycerol release upon ApoL6 overexpression and enhanced lipolysis upon its depletion. The lack of DAG accumulation with ApoL6 overexpression, along with reduced ATGL S406 phosphorylation, suggests a coordinated suppression of overall lipolysis beyond HSL, possibly via altered activation of ATGL, although direct binding to ATGL was not detected. Physiologically, ApoL6 modulates adiposity: its ablation protects against diet-induced obesity and improves glucose/insulin tolerance, whereas adipocyte-specific overexpression increases adiposity, inflammation, hepatic TAG accumulation, and insulin resistance. These data position ApoL6 as a gatekeeper of lipolysis linking feeding status to lipid mobilization and metabolic outcomes.

This work establishes ApoL6 as a lipid droplet protein that inhibits adipocyte lipolysis by directly binding Plin1 and disrupting Plin1–HSL interaction. ApoL6 is induced by feeding, localizes to LD via phosphoinositide interactions, and its gain- or loss-of-function bidirectionally modulates TAG storage, adiposity, and systemic glucose/insulin homeostasis in mice. ApoL6 thus represents a potential therapeutic target for obesity, insulin resistance, and fatty liver disease. Future research should define how ApoL6 influences ATGL activation (e.g., S406 phosphorylation), elucidate regulation of ApoL6 abundance and turnover across nutritional states, assess ApoL6’s roles in non-adipose tissues (e.g., liver in HFD/aging), explore the relevance of human ApoL6 genetic variation to lipid traits, and evaluate pharmacologic strategies to modulate ApoL6–Plin1 interaction.

• The precise mechanism by which ApoL6 overexpression lowers ATGL S406 phosphorylation and suppresses ATGL activity is not resolved; no direct ApoL6–ATGL binding was detected by pull-down despite co-IP within complexes.
• Although ApoL6’s PIP2 binding maps to its C-terminus, the functional importance of specific lipid interactions for LD targeting versus lipolysis inhibition needs further clarification, as lipolysis inhibition was maintained with a PIP2-binding mutant.
• The aP2 promoter can be active in non-adipocyte lineages (e.g., macrophages); an adiponectin promoter TG line was used to corroborate adipocyte specificity, but off-target expression cannot be completely excluded.
• Most mechanistic studies were performed in mouse and cultured adipocytes; translational relevance to human adipose tissue physiology and metabolic disease requires further validation.
• Sex differences were only partially examined; comprehensive sex-specific analyses were limited.
• Sample sizes were modest in some assays; while effects were replicated, broader cohorts would strengthen generalizability.