Deciphering the Role of Lipid Droplets in Cardiovascular Disease: A Report From the 2017 National Heart, Lung, and Blood Institute Workshop.

Lipid droplets (LDs) are distinct and dynamic organelles that affect the health of cells and organs. Much progress has been made in understanding how these structures are formed, how they interact with other cellular organelles, how they are used for storage of triacylglycerol in adipose tissue, and how they regulate lipolysis. Our understanding of the biology of LDs in the heart and vascular tissue is relatively primitive in comparison with LDs in adipose tissue and liver. The National Heart, Lung, and Blood Institute convened a working group to discuss how LDs affect cardiovascular diseases. The goal of the working group was to examine the current state of knowledge on the cell biology of LDs, including current methods to study them in cells and organs and reflect on how LDs influence the development and progression of cardiovascular diseases. This review summarizes the working group discussion and recommendations on research areas ripe for future investigation that will likely improve our understanding of atherosclerosis and heart function.

Qa-SNARE syntaxin 18 mediates lipid droplet fusion with SNAP23 and SEC22B.

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.

The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria.

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.

Perilipin 5 links mitochondrial uncoupled respiration in brown fat to healthy white fat remodeling and systemic glucose tolerance.

Exposure of mice or humans to cold promotes significant changes in brown adipose tissue (BAT) with respect to histology, lipid content, gene expression, and mitochondrial mass and function. Herein we report that the lipid droplet coat protein Perilipin 5 (PLIN5) increases markedly in BAT during exposure of mice to cold. To understand the functional significance of cold-induced PLIN5, we created and characterized gain- and loss-of-function mouse models. Enforcing PLIN5 expression in mouse BAT mimics the effects of cold with respect to mitochondrial cristae packing and uncoupled substrate-driven respiration. PLIN5 is necessary for the maintenance of mitochondrial cristae structure and respiratory function during cold stress. We further show that promoting PLIN5 function in BAT is associated with healthy remodeling of subcutaneous white adipose tissue and improvements in systemic glucose tolerance and diet-induced hepatic steatosis. These observations will inform future strategies that seek to exploit thermogenic adipose tissue as a therapeutic target for type 2 diabetes, obesity, and nonalcoholic fatty liver disease.

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

Lipid droplets in lipogenesis and lipolysis.

Organisms store energy for later use during times of nutrient scarcity. Excess energy is stored as triacylglycerol in lipid droplets during lipogenesis. When energy is required, the stored triacylglycerol is hydrolyzed via activation of lipolytic pathways. The coordination of lipid storage and utilization is regulated by the perilipin family of lipid droplet coat proteins [perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), S3-12, tail-interacting protein of 47 kilodaltons (TIP47), and myocardial lipid droplet protein (MLDP)/oxidative tissues-enriched PAT protein (OXPAT)/lipid storage droplet protein 5 (LSDP5)]. Lipid droplets are dynamic and heterogeneous in size, location, and protein content. The proteins that coat lipid droplets change during lipid droplet biogenesis and are dependent upon multiple factors, including tissue-specific expression and metabolic state (basal vs. lipogenic vs. lipolytic). New data suggest that proteins previously implicated in vesicle trafficking, including Rabs, soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), and motor and cytoskeletal proteins, likely orchestrate the movement and fusion of lipid droplets. Thus, rather than inert cytoplasmic inclusions, lipid droplets are now appreciated as dynamic organelles that are critical for management of cellular lipid stores. That much remains to be discovered is suggested by the recent identification of a novel lipase [adipocyte triglyceride lipase (ATGL)] and lipase regulator [Comparative Gene Identification-58 (CGI-58)], which has led to reconsideration of the decades-old model of lipolysis. Future discovery likely will be driven by the exploitation of model organisms and by human genetic studies.

The PAT family of lipid droplet proteins in heart and vascular cells.

Cytoplasmic lipid droplets (LDs) are organelles in which cells store neutral lipids for use as an energy source in times of need, but they also play important roles in the regulation of key metabolic processes. Although LDs are essential for normal cell function, excess accumulation of intracellular lipid is associated with several metabolic diseases, including obesity, type 2 diabetes, and atherosclerosis. The function of LDs is regulated by their associated proteins, including the members of the PAT family: perilipin, adipophilin/adipose differentiation-related protein, tail-interacting protein 47, S3-12, and OXPAT/myocardial LD protein/lipid-storage droplet protein 5. In this review we discuss the PAT proteins in two cardiovascular contexts: 1) in the atherosclerotic vessel wall, where LDs within macrophage foam cells store cholesteryl esters derived from modified lipoproteins, and 2) in the myocardium, where LDs store fatty acids, the major energy substrate for normal heart function, as triglyceride.

OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.

Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as "OXPAT." Physiologic lipid loading of mouse liver by fasting enriches OXPAT in the lipid droplet tissue fraction. OXPAT resides on lipid droplets with the PAT protein adipophilin in primary cardiomyocytes. Ectopic expression of OXPAT promotes fatty acid-induced triacylglycerol accumulation, long-chain fatty acid oxidation, and mRNAs associated with oxidative metabolism. Consistent with these observations, OXPAT is induced in mouse adipose tissue, striated muscle, and liver by physiological (fasting), pathophysiological (insulin deficiency), pharmacological (peroxisome proliferator-activated receptor [PPAR] agonists), and genetic (muscle-specific PPARalpha overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARgamma agonist treatment induces adipose OXPAT mRNA. Further, adipose OXPAT mRNA negatively correlates with BMI in nondiabetic humans. Our collective data in cells, mice, and humans suggest that OXPAT is a marker for PPAR activation and fatty acid oxidation. OXPAT likely contributes to adaptive responses to the fatty acid burden that accompanies fasting, insulin deficiency, and overnutrition, responses that are defective in obesity and type 2 diabetes.

Metabolic fuel selection: the importance of being flexible.

Studies in genetically engineered mice have shown the importance of cross-talk between organs in the regulation of energy metabolism. In this issue, a careful metabolic characterization of mice with genetic deficiency of the GLUT4 glucose transporter in adipocytes and muscle is reported. These mice compensate for decreased peripheral glucose disposal by increasing hepatic glucose uptake and lipid synthesis as well as by increasing lipid utilization in peripheral tissues. These findings are relevant to humans with type 2 diabetes, in whom a key feature is diminished peripheral glucose disposal.

A proposed model of fat packaging by exchangeable lipid droplet proteins.

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3-12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.

Targeting adipose tissue in the treatment of obesity-associated diabetes.

Adipose tissue regulates numerous physiological processes, and its dysfunction in obese humans is associated with disrupted metabolic homeostasis, insulin resistance and type 2 diabetes mellitus (T2DM). Although several US-approved treatments for obesity and T2DM exist, these are limited by adverse effects and a lack of effective long-term glucose control. In this Review, we provide an overview of the role of adipose tissue in metabolic homeostasis and assess emerging novel therapeutic strategies targeting adipose tissue, including adipokine-based strategies, promotion of white adipose tissue beiging as well as reduction of inflammation and fibrosis.

Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.

Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction.

E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte.

BACKGROUND/PURPOSE: Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism. METHODS: We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes. RESULTS: Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events. CONCLUSION: We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

Population-based risk factors for elevated alanine aminotransferase in a South Texas Mexican-American population.

BACKGROUND AND AIMS: Elevated alanine aminotransferase (ALT >40 IU/mL) is a marker of liver injury but provides little insight into etiology. We aimed to identify and stratify risk factors associated with elevated ALT in a randomly selected population with a high prevalence of elevated ALT (39%), obesity (49%) and diabetes (30%). METHODS: Two machine learning methods, the support vector machine (SVM) and Bayesian logistic regression (BLR), were used to capture risk factors in a community cohort of 1532 adults from the Cameron County Hispanic Cohort (CCHC). A total of 28 predictor variables were used in the prediction models. The recently identified genetic marker rs738409 on the PNPLA3 gene was genotyped using the Sequenom iPLEX assay. RESULTS: The four major risk factors for elevated ALT were fasting plasma insulin level and insulin resistance, increased BMI and total body weight, plasma triglycerides and non-HDL cholesterol, and diastolic hypertension. In spite of the highly significant association of rs738409 in females, the role of rs738409 in the prediction model is minimal, compared to other epidemiological risk factors. Age and drug and alcohol consumption were not independent determinants of elevated ALT in this analysis. CONCLUSIONS: The risk factors most strongly associated with elevated ALT in this population are components of the metabolic syndrome and point to nonalcoholic fatty liver disease (NAFLD). This population-based model identifies the likely cause of liver disease without the requirement of individual pathological diagnosis of liver diseases. Use of such a model can greatly contribute to a population-based approach to prevention of liver disease.

Skeletal muscle triglycerides, diacylglycerols, and ceramides in insulin resistance: another paradox in endurance-trained athletes?

OBJECTIVE: Chronic exercise and obesity both increase intramyocellular triglycerides (IMTGs) despite having opposing effects on insulin sensitivity. We hypothesized that chronically exercise-trained muscle would be characterized by lower skeletal muscle diacylglycerols (DAGs) and ceramides despite higher IMTGs and would account for its higher insulin sensitivity. We also hypothesized that the expression of key skeletal muscle proteins involved in lipid droplet hydrolysis, DAG formation, and fatty-acid partitioning and oxidation would be associated with the lipotoxic phenotype. RESEARCH DESIGN AND METHODS: A total of 14 normal-weight, endurance-trained athletes (NWA group) and 7 normal-weight sedentary (NWS group) and 21 obese sedentary (OBS group) volunteers were studied. Insulin sensitivity was assessed by glucose clamps. IMTGs, DAGs, ceramides, and protein expression were measured in muscle biopsies. RESULTS: DAG content in the NWA group was approximately twofold higher than in the OBS group and ~50% higher than in the NWS group, corresponding to higher insulin sensitivity. While certain DAG moieties clearly were associated with better insulin sensitivity, other species were not. Ceramide content was higher in insulin-resistant obese muscle. The expression of OXPAT/perilipin-5, adipose triglyceride lipase, and stearoyl-CoA desaturase protein was higher in the NWA group, corresponding to a higher mitochondrial content, proportion of type 1 myocytes, IMTGs, DAGs, and insulin sensitivity. CONCLUSIONS: Total myocellular DAGs were markedly higher in highly trained athletes, corresponding with higher insulin sensitivity, and suggest a more complex role for DAGs in insulin action. Our data also provide additional evidence in humans linking ceramides to insulin resistance. Finally, this study provides novel evidence supporting a role for specific skeletal muscle proteins involved in intramyocellular lipids, mitochondrial oxidative capacity, and insulin resistance.

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase.

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.

OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis.

Much knowledge of adipocyte biology has been learned from cell culture models, most notably 3T3-L1 cells. The 3T3-L1 model has several limitations, including the requirement of 2 weeks to generate adipocytes and the waning of adipogenic potential in culture. We have investigated the capacity of OP9 cells, a line of bone marrow-derived mouse stromal cells, to recapitulate adipogenesis. When OP9 cells are given any one of three adipogenic stimuli, they rapidly accumulate triacylglycerol, assume adipocyte morphology, and express adipocyte late marker proteins, including glucose transporter 4 and adiponectin. OP9 cells can differentiate into adipocytes within 2 days. This rapid rate of differentiation allows for the detection of transiently expressed proteins in mature OP9 adipocytes. Adipogenesis in OP9 cells involves the master transcriptional regulator of adipocyte differentiation, peroxisome proliferator-activated receptor gamma (PPARgamma). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins alpha and beta, PPARgamma, sterol-regulatory element binding protein-1, S3-12, and perilipin. OP9 differentiation is not diminished by maintenance in culture at high cell density or by long periods in continuous culture, thereby facilitating the generation of stable cell lines that retain adipogenic potential. Thus, the unique features of OP9 cells will expedite the study of adipocyte biology.

A regulatory role for 1-acylglycerol-3-phosphate-O-acyltransferase 2 in adipocyte differentiation.

Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAG mass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBPbeta and PPARgamma, key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA), are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation.

S3-12, Adipophilin, and TIP47 package lipid in adipocytes.

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.

Lipid rafts and insulin signaling.

Lipid rafts are domains within the plasma membrane that are enriched in cholesterol and lipids with saturated acyl chains. Specific proteins, including many signaling proteins, segregate into lipid rafts, and this process is important for certain signal transduction events in a variety of cell types. Within the past decade, data have emerged from many laboratories that implicate lipid rafts as critical for proper compartmentalization of insulin signaling in adipocytes. A subset of lipid rafts, caveolae, are coated with membrane proteins of the caveolin family. Direct interactions between resident raft proteins (caveolins and flotillin-1) and insulin-signaling molecules may organize these molecules in space and time to ensure faithful transduction of the insulin signal, at least with respect to the glucose-dependent actions of insulin in adipocytes. The in vivo relevance of this model remains to be determined.