White adipose tissue (WAT) is crucial for energy metabolism, storing excess energy as triglycerides (TAG) within lipid droplets (LDs) in adipocytes. During energy deprivation, lipolysis breaks down TAG, releasing fatty acids (FAs) for other organs. This process is tightly regulated according to nutritional status. However, obesity, characterized by increased WAT mass, is a major health concern linked to type 2 diabetes and insulin resistance. Ectopic TAG storage in other tissues further contributes to insulin resistance. Lipolysis involves sequential enzymatic steps catalyzed by adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL). Hormonal signals, such as catecholamines and insulin, regulate lipolysis through post-translational modifications of lipases and LD-associated proteins. Perilipin 1 (Plin1) plays a critical role as an LD-associated protein, acting as a scaffold for the lipolytic complex. Phosphorylation of Plin1, ATGL, and HSL is essential for complex assembly and lipase activation. In the fasted state, hormonal signaling increases protein kinase A (PKA) activity, phosphorylating Plin1, CGI-58 (a cofactor of ATGL), and HSL, promoting lipolysis. In the fed state, insulin reduces PKA activity and activates protein phosphatase 1 (PP-1), suppressing lipolysis. Other LD-associated proteins may also regulate this process, such as GOS2, which inhibits ATGL. This research aimed to understand the role of LD-associated proteins in WAT TAG metabolism by identifying and characterizing ApoL6, an adipose-specific LD-associated protein.

Previous research has extensively studied the lipolytic pathway and the roles of key enzymes and regulatory proteins. The roles of ATGL, HSL, MGL, and Plin1 in lipolysis regulation have been well-established through studies involving genetic knockouts and pharmacological inhibitors. The importance of post-translational modifications, particularly phosphorylation, in modulating lipase activity and protein interactions has been demonstrated. The opposing effects of catecholamines and insulin on lipolysis have been clarified, highlighting the intricate interplay between hormonal signals and lipolytic enzymes. However, the complete picture of LD-associated protein regulation of lipolysis remained incomplete, prompting the current investigation into ApoL6's role.

The study employed a combination of in vitro and in vivo approaches. ApoL6 expression was analyzed in various mouse tissues using RT-qPCR and Northern blotting. Subcellular localization of ApoL6 was determined by immunofluorescence and subcellular fractionation. ApoL6's interaction with phospholipids was investigated using a protein-lipid overlay assay. The functional role of ApoL6 in adipocytes was studied by overexpressing and knocking down ApoL6 in 3T3-L1 cells and human adipocytes differentiated from fibroblasts. Lipolysis rates were measured by assessing FFA and glycerol release. Global ApoL6 knockout (KO) mice and adipose-specific ApoL6 transgenic mice were generated using CRISPR-Cas9 technology and transgenic approaches, respectively. Phenotyping of these mouse models included body weight, WAT mass, adipocyte size, glucose tolerance tests (GTT), insulin tolerance tests (ITT), and serum lipid analysis. The interaction of ApoL6 with other lipolytic proteins was investigated using co-immunoprecipitation (co-IP), GST pull-down assays, and bimolecular fluorescence complementation (BiFC). Statistical analyses were performed using Student's t-tests, multiple t-tests, and two-way ANOVA.

ApoL6 mRNA and protein were highly expressed in adipose tissue, primarily in adipocytes. ApoL6 expression was upregulated upon refeeding and in obese mice, suggesting a link to nutritional status and obesity. ApoL6 localized to LDs, interacting strongly with phosphatidylinositol phosphates (PIs), particularly PI(4,5)P2, via its C-terminal domain. ApoL6 overexpression in adipocytes increased LD size and TAG content, while ApoL6 knockdown had the opposite effect. ApoL6 overexpression inhibited both basal and isoproterenol-stimulated lipolysis in adipocytes, an effect not observed upon inhibition of ATGL and HSL. Global ApoL6 KO mice had reduced WAT mass and were protected from diet-induced obesity and insulin resistance. In contrast, adipose-specific ApoL6 overexpression in transgenic mice led to increased WAT mass, obesity, and insulin resistance. Co-IP and GST pull-down assays revealed that ApoL6 directly interacts with the N-terminal domain of Plin1, preventing Plin1-HSL interaction. BiFC assays confirmed the ApoL6-Plin1 interaction on LDs. The ApoL6-Plin1 interaction was independent of Plin1 phosphorylation. Plin1 knockdown diminished ApoL6's inhibitory effect on lipolysis, highlighting the importance of Plin1 in mediating ApoL6's function. Lower ATGL S406 phosphorylation was observed in WAT of ApoL6 transgenic mice, suggesting a potential effect of ApoL6 on ATGL activity.

This study demonstrates that ApoL6, an adipose-specific LD-associated protein, plays a significant role in regulating lipolysis and energy homeostasis. The findings indicate that ApoL6 functions as a negative regulator of lipolysis, primarily by interfering with the Plin1-HSL interaction. This mechanism is consistent with ApoL6's expression pattern, being upregulated in the fed state and downregulated in the fasted state. The contrasting phenotypes of ApoL6 KO and transgenic mice further support the role of ApoL6 in regulating adiposity and energy metabolism. The observations regarding ATGL phosphorylation suggest a more complex role for ApoL6 in lipolysis, potentially impacting both HSL and ATGL activity. These results have significant implications for understanding the pathogenesis of obesity and related metabolic disorders. The data suggest that ApoL6 could be a potential therapeutic target for obesity and diabetes.

This research reveals ApoL6 as a novel regulator of lipolysis in adipose tissue, acting through direct interaction with Plin1 to inhibit HSL-mediated lipolysis. ApoL6's role in modulating energy balance, adiposity, and insulin sensitivity is demonstrated by contrasting phenotypes of ApoL6 KO and transgenic mice. Future research should explore the detailed molecular mechanisms by which ApoL6 affects ATGL activity and investigate the therapeutic potential of targeting ApoL6 for treating obesity, diabetes, and related metabolic conditions. Further investigation into the regulation of ApoL6 expression and its interaction with other LD-associated proteins will enhance our understanding of adipose tissue function.

The study focused primarily on WAT and its findings may not be entirely generalizable to other adipose tissue depots. Although the use of both mouse and human adipocytes strengthens the findings, further studies using human subjects are necessary for complete validation. The mechanism of ApoL6-mediated ATGL regulation requires further elucidation. The specific role of ApoL6 in the liver, especially under conditions of HFD feeding or aging, deserves further investigation.