LET-767 determines lipid droplet protein targeting and lipid homeostasis.

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.

Vascular ATGL-dependent lipolysis and the activation of cPLA2-PGI2 pathway protect against postprandial endothelial dysfunction.

Adipose triglyceride lipase (ATGL) is involved in lipolysis and displays a detrimental pathophysiological role in cardio-metabolic diseases. However, the organo-protective effects of ATGL-induced lipolysis were also suggested. The aim of this work was to characterize the function of lipid droplets (LDs) and ATGL-induced lipolysis in the regulation of endothelial function. ATGL-dependent LDs hydrolysis and cytosolic phospholipase A2 (cPLA2)-derived eicosanoids production were studied in the aorta, endothelial and smooth muscle cells exposed to exogenous oleic acid (OA) or arachidonic acid (AA). Functional effects of ATGL-dependent lipolysis and subsequent activation of cPLA2/PGI2 pathway were also studied in vivo in relation to postprandial endothelial dysfunction.The formation of LDs was invariably associated with elevated production of endogenous AA-derived prostacyclin (PGI2). In the presence of the inhibitor of ATGL or the inhibitor of cytosolic phospholipase A2, the production of eicosanoids was reduced, with a concomitant increase in the number of LDs. OA administration impaired endothelial barrier integrity in vitro that was further impaired if OA was given together with ATGL inhibitor. Importantly, in vivo, olive oil induced postprandial endothelial dysfunction that was significantly deteriorated by ATGL inhibition, cPLA2 inhibition or by prostacyclin (IP) receptor blockade.In summary, vascular LDs formation induced by exogenous AA or OA was associated with ATGL- and cPLA2-dependent PGI2 production from endogenous AA. The inhibition of ATGL resulted in an impairment of endothelial barrier function in vitro. The inhibition of ATGL-cPLA2-PGI2 dependent pathway resulted in the deterioration of endothelial function upon exposure to olive oil in vivo. In conclusion, vascular ATGL-cPLA2-PGI2 dependent pathway activated by lipid overload and linked to LDs formation in endothelium and smooth muscle cells has a vasoprotective role by counterbalancing detrimental effects of lipid overload on endothelial function.

Cold Ambient Temperature Does Not Alter Subcutaneous Abdominal Adipose Tissue Lipolysis and Blood Flow in Endurance-Trained Cyclists.

This study sought to investigate the effect of cold ambient temperature on subcutaneous abdominal adipose tissue (SCAAT) lipolysis and blood flow during steady-state endurance exercise in endurance-trained cyclists. Ten males (age: 23 ± 3 years; peak oxygen consumption: 60.60 ± 4.84 ml·kg-1·min-1; body fat: 18.4% ± 3.5%) participated in baseline lactate threshold (LT) and peak oxygen consumption testing, two familiarization trials, and two experimental trials. Experimental trials consisted of cycling in COLD (3 °C; 42% relative humidity) and neutral (NEU; 19 °C; 39% relative humidity) temperatures. Exercise consisted of 25 min cycling at 70% LT and 25 min at 90% LT. In situ SCAAT lipolysis and blood flow were measured via microdialysis. Heart rate, core temperature, carbohydrate and fat oxidation, blood glucose, and blood lactate were also measured. Heart rate, core temperature, oxygen consumption, and blood lactate increased with exercise but were not different between COLD and NEU. SCAAT blood flow did not change from rest to exercise or between COLD and NEU. Interstitial glycerol increased during exercise (p < .001) with no difference between COLD and NEU. Fat oxidation increased (p < .001) at the onset of exercise and remained elevated thereafter with no difference between COLD and NEU. Carbohydrate oxidation increased with increasing exercise intensity and was greater at 70% LT in COLD compared to NEU (p = .030). No differences were observed between conditions for any other variable. Cycling exercise increased SCAAT lipolysis but not blood flow. Ambient temperature did not alter SCAAT metabolism, SCAAT blood flow, or fat oxidation in well-trained cyclists, though cold exposure increased whole-body carbohydrate oxidation at lower exercise intensities.

Phillyrin: an adipose triglyceride lipase inhibitor supported by molecular docking, dynamics simulation, and pharmacological validation.

CONTEXT: Adipose triglyceride lipase (ATGL), a key enzyme responsible for lipolysis, catalyzes the first step of lipolysis and converts triglycerides to diacylglycerols and free fatty acids (FFA). Our previous work suggested that phillyrin treatment improves insulin resistance in HFD-fed mice, which was associated with ATGL inhibition. In this study, using docking simulation, we explored the binding pose of phillyrin and atglistatin (a mouse ATGL inhibitor) to ATGL in mouse. From the docking results, the interactions with Ser47 and Asp166 were speculated to have caused phillyrin to inhibit ATGL in mice. Further, molecular dynamics simulation of 100 ns and MM-GBSA were conducted for the protein-ligand complex, which indicated that the system was stable and that phillyrin displayed a better affinity to ATGL than did atglistatin throughout the simulation period. Moreover, the results of pharmacological validation were consistent with those of the in silico simulations. In summary, our study illustrates the potential of molecular docking to accurately predict the binding protein produced by AlphaFold and suggests that phillyrin is a potential small molecule that targets and inhibits ATGL enzymatic activity. METHODS: The ATGL-predicted protein structure, verified by PROCHECK, was determined using AlphaFold. Molecular docking, molecular dynamics simulation, and prime molecular mechanic-generalized born surface area were performed using LigPrep, Desmond, and prime MM-GBSA modules of Schrödinger software release 2021-2, respectively. For pharmacological validation, immunoblotting was performed to assess ATGL protein expression. The fluorescence intensity and glycerol concentration were quantified to evaluate the efficiency of phillyrin in inhibiting ATGL.

p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL.

Adipose tissue lipolysis is mediated by cAMP-protein kinase A (PKA)-dependent intracellular signalling. Here, we show that PKA targets p21-activated kinase 4 (PAK4), leading to its protein degradation. Adipose tissue-specific overexpression of PAK4 in mice attenuates lipolysis and exacerbates diet-induced obesity. Conversely, adipose tissue-specific knockout of Pak4 or the administration of a PAK4 inhibitor in mice ameliorates diet-induced obesity and insulin resistance while enhancing lipolysis. Pak4 knockout also increases energy expenditure and adipose tissue browning activity. Mechanistically, PAK4 directly phosphorylates fatty acid-binding protein 4 (FABP4) at T126 and hormone-sensitive lipase (HSL) at S565, impairing their interaction and thereby inhibiting lipolysis. Levels of PAK4 and the phosphorylation of FABP4-T126 and HSL-S565 are enhanced in the visceral fat of individuals with obesity compared to their lean counterparts. In summary, we have uncovered an important role for FABP4 phosphorylation in regulating adipose tissue lipolysis, and PAK4 inhibition may offer a therapeutic strategy for the treatment of obesity.

Follistatin-like 1 is a myokine regulating lipid mobilization during endurance exercise and recovery.

OBJECTIVE: The aim of this study was to investigate the role of the follistatin-like 1 (Fstl1) and disco-interacting protein 2 homolog A (DIP2a) axis in relation to lipid metabolism during and after endurance exercise and to elucidate the mechanisms underlying the metabolic effects of Fstl1 on adipocytes, considering its regulation by exercise and muscle mass and its link to obesity. METHODS: Twenty-nine sedentary males participated in endurance exercise, and blood samples were collected during and after the exercise. Body composition, Fstl1, glycerol, epinephrine, growth hormone, and atrial natriuretic peptide were measured. 3T3-L1 adipocytes, with or without DIP2a knockdown, were treated with Fstl1 to assess glycerol release, cyclic AMP/cyclic GMP production, and hormone sensitive lipase phosphorylation. The association between DIP2a gene expression levels in human adipose tissues and exercise-induced lipolysis was examined. RESULTS: Fstl1 levels significantly increased during endurance exercise and following recovery, correlating with lean body mass and lipolysis. In 3T3-L1 adipocytes, Fstl1 increased glycerol release, cyclic GMP production, and hormone sensitive lipase activation, but these effects were attenuated by DIP2a knockdown. DIP2a gene expression in human adipose tissues correlated with serum glycerol concentrations during endurance exercise. CONCLUSIONS: Fstl1 is a myokine facilitating lipid mobilization during and after endurance exercise through DIP2a-mediated lipolytic effects in adipocytes.

Acute activation of adipocyte lipolysis reveals dynamic lipid remodeling of the hepatic lipidome.

Adipose tissue is the site of long-term energy storage. During the fasting state, exercise, and cold exposure, the white adipose tissue mobilizes energy for peripheral tissues through lipolysis. The mobilization of lipids from white adipose tissue to the liver can lead to excess triglyceride accumulation and fatty liver disease. Although the white adipose tissue is known to release free fatty acids, a comprehensive analysis of lipids mobilized from white adipocytes in vivo has not been completed. In these studies, we provide a comprehensive quantitative analysis of the adipocyte-secreted lipidome and show that there is interorgan crosstalk with liver. Our analysis identifies multiple lipid classes released by adipocytes in response to activation of lipolysis. Time-dependent analysis of the serum lipidome showed that free fatty acids increase within 30 min of ß3-adrenergic receptor activation and subsequently decrease, followed by a rise in serum triglycerides, liver triglycerides, and several ceramide species. The triglyceride composition of liver is enriched for linoleic acid despite higher concentrations of palmitate in the blood. To further validate that these findings were a specific consequence of lipolysis, we generated mice with conditional deletion of adipose tissue triglyceride lipase exclusively in adipocytes. This loss of in vivo adipocyte lipolysis prevented the rise in serum free fatty acids and hepatic triglycerides. Furthermore, conditioned media from adipocytes promotes lipid remodeling in hepatocytes with concomitant changes in genes/pathways mediating lipid utilization. Together, these data highlight critical role of adipocyte lipolysis in interorgan crosstalk between adipocytes and liver.

Unmasking crucial residues in adipose triglyceride lipase for coactivation with comparative gene identification-58.

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be coactivated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein coactivation on a per-residue level. We identified the region from residues N209-N215 in ATGL as essential for coactivation by CGI-58. ATGL variants with amino acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 coactivation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the coactivation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for coactivation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58-mediated coactivation of ATGL.

The molecular mechanism of obesity: The science behind natural exercise yoga and healthy diets in the treatment of obesity.

The review centers on the scientific evidence underlying obesity, providing a detailed examination of the role of perilipin in this condition. It explores potential causes of obesity and delves into therapeutic approaches involving exercise, yoga, and herbal treatments. The paper discusses natural sources that can contribute to combating obesity and underscores the importance of exercise in a scientific context for overcoming obesity. Additionally, it includes information on herbal ingredients that aid in reducing obesity. The review also examines the impact of exercise type and intensity at various time intervals on muscle development. It elucidates triglyceride hydrolysis through different enzymes and the deposition of fatty acids in adipose tissue. The mechanisms by which alpha/beta hydrolase domain-containing protein 5 (ABHD5) and hormone-sensitive lipase (HSL) target and activate their functions are detailed. The inflammatory response in obesity is explored, encompassing inflammatory markers, lipid storage diseases, and their classification with molecular mechanisms. Furthermore, the hormonal regulation of lipolysis is elaborated upon in the review.

FATP4 deletion in liver cells induces elevation of extracellular lipids via metabolic channeling towards triglycerides and lipolysis.

Evidence from mice with global deletion of fatty-acid transport protein4 (FATP4) indicates its role on ß-oxidation and triglycerides (TG) metabolism. We reported that plasma glycerol and free fatty acids (FA) were increased in liver-specific Fatp4 deficient (L-FATP4-/-) mice under dietary stress. We hypothesized that FATP4 may mediate hepatocellular TG lipolysis. Here, we demonstrated that L-FATP4-/- mice showed an increase in these blood lipids, liver TG, and subcutaneous fat weights. We therefore studied TG metabolism in response to oleate treatment in two experimental models using FATP4-knockout HepG2 (HepKO) cells and L-FATP4-/- hepatocytes. Both FATP4-deificient liver cells showed a significant decrease in ß-oxidation products by ∼30-35% concomitant with marked upregulation of CD36, FATP2, and FATP5 as well as lipoprotein microsomal-triglyceride-transfer protein genes. By using 13C3D5-glycerol, HepKO cells displayed an increase in metabolically labelled TG species which were further increased with oleate treatment. This increase was concomitant with a step-wise elevation of TG in cells and supernatants as well as the secretion of cholesterol very low-density and high-density lipoproteins. Upon analyzing TG lipolytic enzymes, both mutant liver cells showed marked upregulated expression of hepatic lipase, while that of hormone-sensitive lipase and adipose-triglyceride lipase was downregulated. Lipolysis measured by extracellular glycerol and free FA was indeed increased in mutant cells, and this event was exacerbated by oleate treatment. Taken together, FATP4 deficiency in liver cells led to a metabolic shift from ß-oxidation towards lipolysis-directed TG and lipoprotein secretion, which is in line with an association of FATP4 polymorphisms with blood lipids.

Molecular Machinery of Lipid Droplet Degradation and Turnover in Plants.

Lipid droplets (LDs) are important organelles conserved across eukaryotes with a fascinating biogenesis and consumption cycle. Recent intensive research has focused on uncovering the cellular biology of LDs, with emphasis on their degradation. Briefly, two major pathways for LD degradation have been recognized: (1) lipolysis, in which lipid degradation is catalyzed by lipases on the LD surface, and (2) lipophagy, in which LDs are degraded by autophagy. Both of these pathways require the collective actions of several lipolytic and proteolytic enzymes, some of which have been purified and analyzed for their in vitro activities. Furthermore, several genes encoding these proteins have been cloned and characterized. In seed plants, seed germination is initiated by the hydrolysis of stored lipids in LDs to provide energy and carbon equivalents for the germinating seedling. However, little is known about the mechanism regulating the LD mobilization. In this review, we focus on recent progress toward understanding how lipids are degraded and the specific pathways that coordinate LD mobilization in plants, aiming to provide an accurate and detailed outline of the process. This will set the stage for future studies of LD dynamics and help to utilize LDs to their full potential.

Cooperation of acylglycerol hydrolases in neuronal lipolysis.

Genetic and biochemical evidence has established DDHD-domain containing 2 (DDHD2) as the principal triacylglycerol (TAG) hydrolase in neuronal lipolysis of cytosolic lipid droplets. In this issue of Journal of Lipid Research, Hofer et al. report that DDHD2 cooperates with adipose triglyceride lipase, the principal TAG hydrolase in adipose lipolysis, contributing to cytosolic hydrolysis of both TAG and diacylglycerols in murine neuroblastoma cells and primary cortical neurons via different configurations of the lipases. This finding highlights the complexity of cytosolic acylglycerol hydrolysis and raises many new questions in the field of lipid metabolism.

Perilipin 1 Deficiency Prompts Lipolysis in Lipid Droplets and Aggravates the Pathogenesis of Persistent Immune Activation in Drosophila.

Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in lots of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. In this study, we induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. We find that deficiency of Plin1, a key regulator of LDs' reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation.

Adipose METTL14-Elicited N6 -Methyladenosine Promotes Obesity, Insulin Resistance, and NAFLD Through Suppressing ß Adrenergic Signaling and Lipolysis.

White adipose tissue (WAT) lipolysis releases free fatty acids as a key energy substance to support metabolism in fasting, cold exposure, and exercise. Atgl, in concert with Cgi-58, catalyzes the first lipolytic reaction. The sympathetic nervous system (SNS) stimulates lipolysis via neurotransmitter norepinephrine that activates adipocyte ß adrenergic receptors (Adrb1-3). In obesity, adipose Adrb signaling and lipolysis are impaired, contributing to pathogenic WAT expansion; however, the underling mechanism remains poorly understood. Recent studies highlight importance of N6 -methyladenosine (m6A)-based RNA modification in health and disease. METTL14 heterodimerizes with METTL3 to form an RNA methyltransferase complex that installs m6A in transcripts. Here, this work shows that adipose Mettl3 and Mettl14 are influenced by fasting, refeeding, and insulin, and are upregulated in high fat diet (HFD) induced obesity. Adipose Adrb2, Adrb3, Atgl, and Cgi-58 transcript m6A contents are elevated in obesity. Mettl14 ablation decreases these transcripts' m6A contents and increases their translations and protein levels in adipocytes, thereby increasing Adrb signaling and lipolysis. Mice with adipocyte-specific deletion of Mettl14 are resistant to HFD-induced obesity, insulin resistance, glucose intolerance, and nonalcoholic fatty liver disease (NAFLD). These results unravel a METTL14/m6A/translation pathway governing Adrb signaling and lipolysis. METTL14/m6A-based epitranscriptomic reprogramming impairs adipose Adrb signaling and lipolysis, promoting obesity, NAFLD, and metabolic disease.

The Potential Roles of Transacylation in Intracellular Lipolysis and Related Qssa Approximations.

Fatty acids (FAs) are crucial energy metabolites, signalling molecules, and membrane building blocks for a wide range of organisms. Adipose triglyceride lipase (ATGL) is the first and presumingly most crucial regulator of FA release from triacylglycerols (TGs) stored within cytosolic lipid droplets. However, besides the function of releasing FAs by hydrolysing TGs into diacylglycerols (DGs), ATGL also promotes the transacylation reaction of two DG molecules into one TG and one monoacylglycerol molecule. To date, it is unknown whether DG transacylation is a coincidental byproduct of ATGL-mediated lipolysis or whether it is physiologically relevant. Experimental evidence is scarce since both, hydrolysis and transacylation, rely on the same active site of ATGL and always occur in parallel in an ensemble of molecules. This paper illustrates the potential roles of transacylation. It shows that, depending on the kinetic parameters but also on the state of the hydrolytic machinery, transacylation can increase or decrease downstream products up to 80% respectively 30%. We provide an extensive asymptotic analysis including quasi-steady-state approximations (QSSA) with higher order correction terms and provide numerical simulation. We also argue that when assessing the validity of QSSAs one should include parameter sensitivity derivatives. Our results suggest that the transacylation function of ATGL is of biological relevance by providing feedback options and altogether stability to the lipolytic machinery in adipocytes.

Exercise Equals the Mobilization of Visceral versus Subcutaneous Adipose Fatty Acid Molecules in Fasted Rats Associated with the Modulation of the AMPK/ATGL/HSL Axis.

Combining exercise with fasting is known to boost fat mass-loss, but detailed analysis on the consequential mobilization of visceral and subcutaneous WAT-derived fatty acids has not been performed. In this study, a subset of fasted male rats (66 h) was submitted to daily bouts of mild exercise. Subsequently, by using gas chromatography-flame ionization detection, the content of 22 fatty acids (FA) in visceral (v) versus subcutaneous (sc) white adipose tissue (WAT) depots was compared to those found in response to the separate events. Findings were related to those obtained in serum and liver samples, the latter taking up FA to increase gluconeogenesis and ketogenesis. Each separate intervention reduced scWAT FA content, associated with increased levels of adipose triglyceride lipase (ATGL) protein despite unaltered AMP-activated protein kinase (AMPK) Thr172 phosphorylation, known to induce ATGL expression. The mobility of FAs from vWAT during fasting was absent with the exception of the MUFA 16:1 n-7 and only induced by combining fasting with exercise which was accompanied with reduced hormone sensitive lipase (HSL) Ser563 and increased Ser565 phosphorylation, whereas ATGL protein levels were elevated during fasting in association with the persistently increased phosphorylation of AMPK at Thr172 both during fasting and in response to the combined intervention. As expected, liver FA content increased during fasting, and was not further affected by exercise, despite additional FA release from vWAT in this condition, underlining increased hepatic FA metabolism. Both fasting and its combination with exercise showed preferential hepatic metabolism of the prominent saturated FAs C:16 and C:18 compared to the unsaturated FAs 18:1 n-9 and 18:2 n-6:1. In conclusion, depot-specific differences in WAT fatty acid molecule release during fasting, irrelevant to their degree of saturation or chain length, are mitigated when combined with exercise, to provide fuel to surrounding organs such as the liver which is correlated with increased ATGL/ HSL ratios, involving AMPK only in vWAT.

Endothelial-derived FABP4 constitutes the majority of basal circulating hormone and regulates lipolysis-driven insulin secretion.

Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with obesity and metabolic pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with Fabp4 deletion in cells known to express the gene - adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) - to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was not significantly reduced in Adipo-KO mice, whereas Endo-KO mice showed ~87% reduction versus WT controls. In contrast, Adipo-KO mice exhibited ~62% decreased induction of FABP4 responses to lipolysis, while Endo-KO mice showed only mildly decreased induction, indicating that adipocytes are the main source of increases in FABP4 during lipolysis. We did not detect any myeloid contribution to circulating FABP4. Surprisingly, despite the nearly intact induction of FABP4, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis.

A comprehensive mechanistic model of adipocyte signaling with layers of confidence.

Adipocyte signaling, normally and in type 2 diabetes, is far from fully understood. We have earlier developed detailed dynamic mathematical models for several well-studied, partially overlapping, signaling pathways in adipocytes. Still, these models only cover a fraction of the total cellular response. For a broader coverage of the response, large-scale phosphoproteomic data and systems level knowledge on protein interactions are key. However, methods to combine detailed dynamic models with large-scale data, using information about the confidence of included interactions, are lacking. We have developed a method to first establish a core model by connecting existing models of adipocyte cellular signaling for: (1) lipolysis and fatty acid release, (2) glucose uptake, and (3) the release of adiponectin. Next, we use publicly available phosphoproteome data for the insulin response in adipocytes together with prior knowledge on protein interactions, to identify phosphosites downstream of the core model. In a parallel pairwise approach with low computation time, we test whether identified phosphosites can be added to the model. We iteratively collect accepted additions into layers and continue the search for phosphosites downstream of these added layers. For the first 30 layers with the highest confidence (311 added phosphosites), the model predicts independent data well (70-90% correct), and the predictive capability gradually decreases when we add layers of decreasing confidence. In total, 57 layers (3059 phosphosites) can be added to the model with predictive ability kept. Finally, our large-scale, layered model enables dynamic simulations of systems-wide alterations in adipocytes in type 2 diabetes.

The (social) lives, deaths, and biophysical phases of lipid droplets.

Lipid droplets (LDs) are major lipid storage organelles, sequestering energy-rich triglycerides and serving as nutrient sinks for cellular homeostasis. Observed for over a century but generally ignored, LDs are now appreciated to play key roles in organismal physiology and disease. They also form numerous functional contacts with other organelles. Here, we highlight recent studies examining LDs from distinct perspectives of their life cycle: their biogenesis, "social" life as they interact with other organelles, and deaths via lipolysis or lipophagy. We also discuss recent work showing how changes in LD lipid content alter the biophysical phases of LD lipids, and how this may fine-tune the LD protein landscape and ultimately LD function.

Sympathetic tone dictates the impact of lipolysis on FABP4 secretion.

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by ß-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGLAdpKO). Here, we find that upon activation of ß-adrenergic receptors in vivo, ATGLAdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGLfl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGLAdpKO mice was indeed from the adipocytes. ATGLAdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGLAdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.