Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.     

Genetic deletion or pharmacologic inhibition of the Nlrp3 inflammasome did not ameliorate experimental NASH

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.     

Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase

The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport.     

Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.     

The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation

Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.     

Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

Background

Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1β and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1β has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway.

Principal Findings

Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1β from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1β was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1β secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1β secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization.

Conclusions

The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.     

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Background & Aims

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).

Methods & Results

Ldlr^−/− mice were transplanted with wild-type (Wt), Cd36^−/− or Msr1^−/− bone marrow and fed a Western diet for 3months. Cd36^−/−- and Msr1^−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36^−/−- and Msr1^−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.

Conclusion

CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.     

Adipose tissue conditioned media support macrophage lipid-droplet biogenesis by interfering with autophagic flux

Obesity promotes the biogenesis of adipose tissue (AT) foam cells (FC), which contribute to AT insulin resistance. Autophagy, an evolutionarily-conserved house-keeping process, was implicated in cellular lipid handling by either feeding and/or degrading lipid-droplets (LDs). We hypothesized that beyond phagocytosis of dead adipocytes, AT-FC biogenesis is supported by the AT microenvironment by regulating autophagy. Non-polarized (“M0”) RAW264.7 macrophages exposed to AT conditioned media (AT-CM) exhibited a markedly enhanced LDs biogenesis rate compared to control cells (8.3 Vs 0.3 LDs/cells/h, p < 0.005). Autophagic flux was decreased by AT-CM, and fluorescently following autophagosomes over time revealed ~ 20% decline in new autophagic vesicles' formation rate, and 60–70% decrease in autophagosomal growth rate, without marked alternations in the acidic lysosomal compartment. Suppressing autophagy by either targeting autophagosome formation (pharmacologically, with 3-methyladenine or genetically, with Atg12 ± Atg7-siRNA), decreased the rate of LD formation induced by oleic acid. Conversely, interfering with late autophago-lysosomal function, either pharmacologically with bafilomycin-A1, chloroquine or leupeptin, enhanced LD formation in macrophages without affecting LD degradation rate. Similarly enhanced LD biogenesis rate was induced by siRNA targeting Lamp-1 or the V-ATPase. Collectively, we propose that secreted products from AT interrupt late autophagosome maturation in macrophages, supporting enhanced LDs biogenesis and AT-FC formation, thereby contributing to AT dysfunction in obesity.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.     

Hepatocytes Deficient in Nuclear Envelope Protein Lamina-associated Polypeptide 1 are an Ideal Mammalian System to Study Intranuclear Lipid Droplets

Lipid droplets (LDs) are generally considered to be synthesized in the ER and utilized in the cytoplasm. However, LDs have been observed inside nuclei in some cells, although recent research on nuclear LDs has focused on cultured cell lines. To better understand nuclear LDs that occur in vivo, here we examined LDs in primary hepatocytes from mice following depletion of the nuclear envelope protein lamina-associated polypeptide 1 (LAP1). Microscopic image analysis showed that LAP1-depleted hepatocytes contain frequent nuclear LDs, which differ from cytoplasmic LDs in their associated proteins. We found type 1 nucleoplasmic reticula, which are invaginations of the inner nuclear membrane, are often associated with nuclear LDs in these hepatocytes. Furthermore, in vivo depletion of the nuclear envelope proteins lamin A and C from mouse hepatocytes led to severely abnormal nuclear morphology, but significantly fewer nuclear LDs than were observed upon depletion of LAP1. In addition, we show both high-fat diet feeding and fasting of mice increased cytoplasmic lipids in LAP1-depleted hepatocytes but reduced nuclear LDs, demonstrating a relationship of LD formation with nutritional state. Finally, depletion of microsomal triglyceride transfer protein did not change the frequency of nuclear LDs in LAP1-depleted hepatocytes, suggesting that it is not required for the biogenesis of nuclear LDs in these cells. Together, these data show that LAP1-depleted hepatocytes represent an ideal mammalian system to investigate the biogenesis of nuclear LDs and their partitioning between the nucleus and cytoplasm in response to changes in nutritional state and cellular metabolism in vivo.     

Hydroxysteroid dehydrogenase family proteins on lipid droplets through bacteria, C. elegans, and mammals

Lipid droplets (LDs) are the main fat storing sites in almost all species from bacteria to humans. The perilipin family has been found as LD proteins in mammals, Drosophila, and a couple of slime molds, but no bacterial LD proteins containing sequence conservation were identified. In this study, we reported that the hydroxysteroid dehydrogenase (HSD) family was found on LDs across all organisms by LD proteomic analysis. Imaging experiments confirmed LD targeting of three representative HSD proteins including ro01416 in RHA1, DHS-3 in C. elegans, and 17β-HSD11 in human cells. In C. elegans, 17β-HSD11 family proteins (DHS-3, DHS-4 and DHS-19) were localized on LDs in distinct tissues. In intestinal cells of C. elegans, DHS-3 targeted to cytoplasmic LDs, while DHS-9 labeled nuclear LDs. Furthermore, the N-terminal hydrophobic domains of 17β-HSD11 family were necessary for their targeting to LDs. Last, 17β-HSD11 family proteins induced LD aggregation, and deletion of DHS-3 in C. elegans caused lipid decrease. Independent of their presumptive catalytic sites, 17β-HSD11 family proteins regulated LD dynamics and lipid metabolism through affecting the LD-associated ATGL, which was conserved between C. elegans and humans. Together, these findings for HSDs provide a new insight not only into the mechanistic studies of the dynamics and functions of LDs in multiple organisms, but also into understanding the evolutionary history of the organelle.     

Polyunsaturated fatty acids promote the rapid fusion of lipid droplets in Caenorhabditis elegans

Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LD fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here, we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal β-oxidation pathway in Caenorhabditis elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show that dysfunction of peroxisomal β-oxidation results in the accumulation of long-chain fatty acid-CoA and phosphocholine, which may activate the sterol-binding protein 1/sterol regulatory element–binding protein to promote gLD formation. Furthermore, we found that inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids (PUFAs) with three or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or phosphocholine bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal β-oxidation–defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.     

The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis

The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.     

Identification and functional characterization of a lipid droplet protein CtLDP1 from an oleaginous yeast Candida tropicalis SY005

Proteins residing in lipid droplets (LDs) of organisms exhibit diverse physiological roles. Since the LD proteins of yeasts are largely unexplored, we have identified a putative LD protein gene, CtLDP1 in the oleaginous yeast Candida tropicalis SY005 and characterized its function. The increased lipid accumulation in SY005 could be correlated with enhanced (~2.67-fold) expression of the CtLDP1 after low-nitrogen stress. The N-terminal transmembrane domain similar to perilipin proteins and the amphipathic α-helices predicted in silico, presumably aid in targeting the CtLDP1 to LD membranes. Heterologous expression of CtLDP1-mCherry fusion in Saccharomyces cerevisiae revealed localization in LDs, yet the expression of CtLDP1 did not show significant effect on LD formation in transformed cells. Molecular docking showed favourable interactions of the protein with sterol class of molecules, but not with triacylglycerol (TAG); and this was further experimentally verified by co-localization of the mCherry-tagged protein in TAG-deficient (but steryl ester containing) LDs. While oleic acid supplementation caused coalescence of LDs into supersized ones (average diameter = 1.19 ± 0.12 μm; n = 160), this effect was suppressed due to CtLDP1 expression, and the cells mostly exhibited numerous smaller LDs (average diameter = 0.46 ± 0.05 μm; n = 160). Moreover, CtLDP1 expression in pet10Δ knockout strain of S. cerevisiae restored multiple LD formation, indicating functional complementation of the protein. Overall, this study documents functional characterization of an LD-stabilizing protein from an oleaginous strain of Candida genus for the first time, and provides insights on the characteristics of LD proteins in oleaginous yeasts for future metabolic engineering.     

Lipolysis and lipophagy play individual and interactive roles in regulating triacylglycerol and cholesterol homeostasis and mitochondrial form in zebrafish

Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl^?/?; AKO fish) or lysosomal acid lipase (lal^?/?; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.     

Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme

Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophylic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen-fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), α-tocopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids, whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location, these structures represent a novel sub-organelle in cyanobacteria.     

The MAP1-LC3 conjugation system is involved in lipid droplet formation

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.