Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma.

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis.

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

2-Bromopalmitate depletes lipid droplets to inhibit viral replication.

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.

Peridroplet mitochondria are associated with the severity of MASLD and the prevention of MASLD by diethyldithiocarbamate.

Mitochondria can contact lipid droplets (LDs) to form peridroplet mitochondria (PDM) which trap fatty acids in LDs by providing ATP for triglyceride synthesis and prevent lipotoxicity. However, the role of PDM in metabolic dysfunction associated steatotic liver disease (MASLD) is not clear. Here, the features of PDM in dietary MASLD models with different severity in mice were explored. Electron microscope photographs show that LDs and mitochondria rarely come into contact with each other in normal liver. In mice fed with high-fat diet, PDM can be observed in the liver as early as the beginning of steatosis in hepatocytes. For the first time, we show that PDM in mouse liver varies with the severity of MASLD. PDM and cytosolic mitochondria were isolated from the liver tissue of MASLD and analyzed by quantitative proteomics. Compared with cytosolic mitochondria, PDM have enhanced mitochondrial respiration and ATP synthesis. Diethyldithiocarbamate (DDC) alleviates choline-deficient, L-amino acid-defined diet-induced MASLD, while increases PDM in the liver. Similarly, DDC promotes the contact of mitochondria-LDs in steatotic C3A cells in vitro. Meanwhile, DDC promotes triglyceride synthesis and improves mitochondrial dysfunction in MASLD. In addition, DDC upregulates perilipin 5 both in vivo and in vitro, which is considered as a key regulator in PDM formation. Knockout of perilipin 5 inhibits the contact of mitochondria-LDs induced by DDC in C3A cells. These results demonstrate that PDM might be associated with the progression of MASLD and the prevention of MASLD by DDC.

Lycium barbarum polysaccharide ameliorates the accumulation of lipid droplets in adipose tissue via an ATF6/SIRT1-dependent mechanism.

Lipid droplets (LDs) are dynamic organelles that store neutral lipids and are closely linked to obesity. Previous studies have suggested that Lycium barbarum polysaccharide (LBP) supplements can ameliorate obesity, but the underlying mechanisms remain unclear. In this study, we hypothesize that LBP alleviates LD accumulation in adipose tissue (AT) by inhibiting fat-specific protein 27 (Fsp27) through an activating transcription factor-6 (ATF6)/small-molecule sirtuin 1 (SIRT1)-dependent mechanism. LD accumulation in AT is induced in high-fat diet (HFD)-fed mice, and differentiation of 3T3-L1 preadipocytes (PAs) is induced. The ability of LBP to alleviate LD accumulation and the possible underlying mechanism are then investigated both in vivo and in vitro. The influences of LBP on the expressions of LD-associated genes ( ATF6 and Fsp27) are also detected. The results show that HFD and PA differentiation markedly increase LD accumulation in ATs and adipocytes, respectively, and these effects are markedly suppressed by LBP supplementation. Furthermore, LBP significantly activates SIRT1 and decreases ATF6 and Fsp27 expressions. Interestingly, the inhibitory effects of LBP are either abolished or exacerbated when ATF6 is overexpressed or silenced, respectively. Furthermore, SIRT1 level is transcriptionally regulated by LBP through opposite actions mediated by ATF6. Collectively, our findings suggest that LBP supplementation alleviates obesity by ameliorating LD accumulation, which might be partially mediated by an ATF6/SIRT1-dependent mechanism.

Inhibition of Drp1-Filamin Protein Complex Prevents Hepatic Lipid Droplet Accumulation by Increasing Mitochondria-Lipid Droplet Contact.

Lipid droplet (LD) accumulation in hepatocytes is one of the major symptoms associated with fatty liver disease. Mitochondria play a key role in catabolizing fatty acids for energy production through ß-oxidation. The interplay between mitochondria and LD assumes a crucial role in lipid metabolism, while it is obscure how mitochondrial morphology affects systemic lipid metabolism in the liver. We previously reported that cilnidipine, an already existing anti-hypertensive drug, can prevent pathological mitochondrial fission by inhibiting protein-protein interaction between dynamin-related protein 1 (Drp1) and filamin, an actin-binding protein. Here, we found that cilnidipine and its new dihydropyridine (DHP) derivative, 1,4-DHP, which lacks Ca2+ channel-blocking action of cilnidipine, prevent the palmitic acid-induced Drp1-filamin interaction, LD accumulation and cytotoxicity of human hepatic HepG2 cells. Cilnidipine and 1,4-DHP also suppressed the LD accumulation accompanied by reducing mitochondrial contact with LD in obese model and high-fat diet-fed mouse livers. These results propose that targeting the Drp1-filamin interaction become a new strategy for the prevention or treatment of fatty liver disease.

Lipid Droplets Are Beneficial for Rabies Virus Replication by Facilitating Viral Budding.

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been reported to play a role in pathogenesis of several viruses. However, their role in RABV infection remains unclear. Here, we initially found that RABV infection upregulated LD production in multiple cells and mouse brains. After treatment with atorvastatin, a specific inhibitor of LDs, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhanced the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglyceride synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication, and their biogenesis is regulated via the NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but their role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier for transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

Cinnamaldehyde affects lipid droplets metabolism after adipogenic differentiation of C2C12 cells.

BACKGROUND: Based on our previous research conducted on cinnamaldehyde (CA) exhibiting its ability to improve the growth performance of fattening pigs and the adipogenesis induction model of C2C12 cells constructed in our laboratory, we explored the effects of CA on the generation and development of lipid droplets (LDs) in adipogenic differentiated C2C12 cells. METHODS AND RESULTS: C2C12 cells were treated with either 0.4 mM or 0.8 mM CA. BODIPY staining and triglyceride measurements were conducted to observe the morphology of LDs, and Western blotting was used to measure the expression of their metabolism-related proteins. The results showed that the average number of LDs in the CA treatment groups was more than the control group (P < 0.05), whereas the average LD size and triglyceride content decreased (P < 0.05). Compared with the control group, the expression levels of fusion-related genes in the LDs of the CA treatment group significantly decreased, while decomposition-related genes and autophagy-related genes in the LDs in C2C12 cells significantly increased (P < 0.01). CONCLUSION: Cinnamaldehyde promoted the decomposition and autophagy of lipid droplets in C2C12 cells and inhibited the fusion of lipid droplets.

Lipid droplets can promote drug accumulation and activation.

Genetic screens in cultured human cells represent a powerful unbiased strategy to identify cellular pathways that determine drug efficacy, providing critical information for clinical development. We used insertional mutagenesis-based screens in haploid cells to identify genes required for the sensitivity to lasonolide A (LasA), a macrolide derived from a marine sponge that kills certain types of cancer cells at low nanomolar concentrations. Our screens converged on a single gene, LDAH, encoding a member of the metabolite serine hydrolase family that is localized on the surface of lipid droplets. Mechanistic studies revealed that LasA accumulates in lipid droplets, where it is cleaved into a toxic metabolite by LDAH. We suggest that selective partitioning of hydrophobic drugs into the oil phase of lipid droplets can influence their activation and eventual toxicity to cells.

Dietary Carbohydrates and Fat Induce Distinct Surfactant Alterations in Mice.

Obesity and type 2 diabetes are nutrition-related conditions associated with lung function impairment and pulmonary diseases; however, the underlying pathomechanisms are incompletely understood. Pulmonary surfactant is essential for lung function, and surfactant synthesis by AT2 (alveolar epithelial type 2) cells relies on nutrient uptake. We hypothesized that dietary amounts of carbohydrates or fat affect surfactant homeostasis and composition. Feeding mice a starch-rich diet (StD), sucrose-rich diet (SuD), or fat-rich diet (FaD) for 30 weeks resulted in hypercholesterolemia and hyperinsulinemia compared with a fiber-rich control diet. In SuD and FaD groups, lung mechanic measurements revealed viscoelastic changes during inspiration, indicating surfactant alterations, and interfacial adsorption of isolated surfactant at the air-liquid interface was decreased under FaD. The composition of characteristic phospholipid species was modified, including a shift from dipalmitoyl-phosphatidylcholine (PC16:0/16:0) to palmitoyl-palmitoleoyl-phosphatidylcholine (PC16:0/16:1) in response to carbohydrates and decreased myristic acid-containing phosphatidylcholine species (PC14:0/14:0; PC16:0/14:0) on excess fat intake, as well as higher palmitoyl-oleoyl-phosphatidylglycerol (PG16:0/18:1) and palmitoyl-linoleoyl-phosphatidylglycerol (PG16:0/18:2) fractions in StD, SuD, and FaD groups than in the control diet. Moreover, mRNA expression levels of surfactant synthesis-related proteins within AT2 cells were altered. Under the StD regimen, AT2 cells showed prominent lipid accumulations and smaller lamellar bodies. Thus, in an established mouse model, distinct diet-related surfactant alterations were subtle, yet detectable, and may become challenging under conditions of reduced respiratory capacity. Dietary fat was the only macronutrient significantly affecting surfactant function. This warrants future studies examining alimentary effects on lung surfactant, with special regard to pulmonary complications in obesity and type 2 diabetes.

Physiological and pathophysiological concentrations of fatty acids induce lipid droplet accumulation and impair functional performance of tissue engineered skeletal muscle.

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.

PPAR gamma agonist leriglitazone improves frataxin-loss impairments in cellular and animal models of Friedreich Ataxia.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence in diabetes. The underlying pathophysiological mechanism of FRDA, driven by a significantly decreased expression of frataxin (FXN), involves increased oxidative stress, reduced activity of enzymes containing iron­sulfur clusters (ISC), defective energy production, calcium dyshomeostasis, and impaired mitochondrial biogenesis, leading to mitochondrial dysfunction. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcriptional factor playing a key role in mitochondrial function and biogenesis, fatty acid storage, energy metabolism, and antioxidant defence. It has been previously shown that the PPARγ/PPARγ coactivator 1 alpha (PGC-1α) pathway is dysregulated when there is frataxin deficiency, thus contributing to FRDA pathogenesis and supporting the PPARγ pathway as a potential therapeutic target. Here we assess whether MIN-102 (INN: leriglitazone), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and animal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+ exchanger (NCLX), resulting in an improvement of mitochondrial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase frataxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid ß-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. The results reinforced the different tissue requirement in FRDA and the pleiotropic effects of leriglitazone that could be a promising therapy for FRDA.

Identification of a new autophagy inhibitor targeting lipid droplets in vascular endothelial cells.

Autophagy of vascular endothelial cells (VECs) plays an important role in maintaining vascular homeostasis. Lipid droplets (LDs) are organelles that can be formed in response to various stimuli, including excessive lipid or various stresses. LDs sequester toxic lipids, thereby preventing lipotoxic cell damage and have a complex relationship with autophagy. In the previous study, we identified a novel Grp94 inhibitor HCP1 inhibited apoptosis in VECs. Here we found that HCP1 targeted LDs and promoted the accumulation of LDs in VECs. Our results showed that HCP1 upregulated the protein levels of autophagy-related proteins. We demonstrated that HCP1 upregulated the number of LDs and suppressed autophagy by inhibiting Grp94. Therefore, we provided HCP1 as a new VECs autophagy inhibitor targeting LDs, which might be a potential compound in the treatment of VECs autophagy related vascular diseases.

Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

Atorvastatin Enhances Foam Cell Lipophagy and Promotes Cholesterol Efflux Through the AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

ABSTRACT: Foam cells are the main pathological components of atherosclerosis. Therapies reducing foam cell formation can effectively prevent atherosclerotic diseases and cardiovascular events. Beyond lowering plasma cholesterol levels, the pleiotropic functions of statins in atherosclerosis have not been fully elucidated. In the present study, atorvastatin reduced cholesterol content and increased cholesterol efflux from foam cells in a concentration-dependent manner. Atorvastatin (10 µM) inhibited foam cell formation within 48 hours. Furthermore, we found that atorvastatin inhibited foam cell formation by promoting lipophagy, which was manifested by increased autophagy-related gene 5 (Atg5) expression, elevated ratio of microtubule-associated protein1 light chain 3 (LC3) II to LC3I, reduced p62 expression, and increased LC3 and lipid droplets colocalization in foam cells treated with atorvastatin. The autophagy inducer, rapamycin (Rap), did not increase the lipophagy enhancement effect of atorvastatin, but the autophagy inhibitor, 3-methyladenine, suppressed the effect of atorvastatin on Atg5 expression and the LC3II/LC3I ratio, as well as the increased p62 expression, suppressed lipophagy, attenuated cholesterol efflux and increased cholesterol content in foam cells. Further analysis revealed that atorvastatin promoted lipophagy by upregulating adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation, and downregulating mammalian target of rapamycin phosphorylation, whereas the AMPK inhibiter, compound C, attenuated these effects. In conclusion, atorvastatin reduced lipid accumulation and promoted cholesterol efflux by enhancing lipophagy in foam cells and thereby inhibited foam cell formation. The enhanced lipophagy of foam cells was exerted through the AMPK/mammalian target of rapamycin signaling pathway.

Effects of progesterone on the lipolysis of lipid droplets and prostaglandin E2 synthesis in murine cervical epithelial cells.

Previous studies demonstrated that progesterone (P4) can promote prostaglandin (PG) E2 production; however, how P4 mediates the synthesis of PGE2 remains unclear. In this study, cervical epithelial cells from mice during the follicular phase were cultured invitro and treated with different concentrations of P4 (5, 10, and 20nM). The results of the present study suggest that treatment of murine cervical epithelial cells with 10nM P4 for 24h contributed to: (1) significantly increased expression of protein kinase A (PKA), cytosolic phospholipase A2 (cPLA2) and PGE synthase (PGES)-1; (2) higher phosphorylated (p-) to total extracellular signal-regulated kinase (ERK) 1/2 and hormone-sensitive lipase (HSL) ratios; (3) a significant decrease in the number of lipid droplets (LDs) and fatty acid content within LDs in epithelial cells; and (4) enhanced arachidonic acid and PGE2 levels in cells compared with the control (0nM P4) group (P<0.01 for all findings). In contrast, the PKA inhibitor H89 contributed to significantly decreased cPLA2, PGES-1 and HSL expression, ERK1/2 phosphorylation and arachidonic acid and PGE2 levels, even in the presence of P4. These data show that P4 can act via the PKA/ERK1/2 pathway to stimulate lipolysis of triacylglycerol in the LD core and degradation of phospholipid in the LD membrane to promote PGE2 synthesis in murine cervical epithelial cells.

Impact of short-term high-fat feeding on lipid droplet content in mouse oocytes.

Mature mammalian oocytes contain lipid droplets (LDs), which are neutral lipid storage organelles critically important for energy metabolism. In mice, maternal obesity, induced by long-term (> 3 months) high-fat feeding, contributes to the accumulation of LDs in mature oocytes. However, few studies have investigated the influence of short-term high-fat feeding on LD content. In this study, we demonstrated that 3 weeks of high-fat feeding is sufficient to increase LD content and intracellular triacylglycerol levels. Using a two-step centrifugation technique to release LDs into the perivitelline space, we found that short-term high-fat feeding increased the level of LDs in MII oocytes and that 3 days of high-fat feeding were sufficient to increase efficiency of LD release. Collectively, our study suggests that short-term high fat feeding can have a higher impact on lipid metabolism during oocyte maturation.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 1. Production, milk fatty acid profile, and nutrient digestibility.

The objectives of this experiment were to determine the effects of increased diet fermentability and polyunsaturated fatty acids (FA) with or without supplemental 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), isoacids (IA; isobutyrate, 2-methylbutyrate, isovalerate, and valerate) or the combination of these on milk fat depression (MFD). Ten Holstein cows (194 ± 58 DIM, 691 ± 69 kg BW, 28 ± 5 kg milk yield) were used in a replicated 5 × 5 Latin square design. Treatments included a high-forage control diet (HF-C), a low-forage control diet (LF-C) causing MFD by increasing starch and decreasing neutral detergent fiber (NDF), the LF-C diet supplemented with HMTBa at 0.11% (28 g/d), the LF-C diet supplemented with IA at 0.24% of dietary dry matter (60 g/d), and the LF-C diet supplemented with HMTBa and IA. Preplanned contrasts were used to compare HF-C versus LF-C and to examine the main effects of HMTBa or IA and their interactions within the LF diets. Dry matter intake was greater for LF-C versus HF-C, but milk yield remained unchanged. The LF-C diet decreased milk fat yield (0.87 vs. 0.98 kg/d) but increased protein yield compared with HF-C. As a result, energy-corrected milk was lower (28.5 vs. 29.6 kg/d) for LF-C versus HF-C. Although the concentration of total de novo synthesized FA in milk fat was not affected, some short- and medium-chain FA were lower for LF-C versus HF-C, but the concentrations of C18 trans-10 isomers were not different. Total-tract NDF apparent digestibility was numerically lower (42.4 vs. 45.6%) for LF-C versus HF-C. As the main effects, the decrease in milk fat yield observed in LF-C was alleviated by supplementation of HMTBa through increasing milk yield without altering milk fat content and by IA through increasing milk fat content without altering milk yield so that HMTBa or IA, as the main effects, increased milk fat yield within the LF diets. However, interactions for milk fat yield and ECM were observed between HMTBa and IA, suggesting no additive effect when used in combination. Minimal changes were found on milk FA profile when HMTBa was provided. However, de novo synthesized FA increased for IA supplementation. We detected no main effect of HMTBa, IA, and interaction between those on total-tract NDF digestibility. In conclusion, the addition of HMTBa and IA to a low-forage and high-starch diet alleviated moderate MFD. Although the mechanism by which MFD was alleviated was different between HMTBa and IA, no additive effects of the combination were observed on milk fat yield and ECM.

Cellular Mechanism Underlying Highly-Active or Antiretroviral Therapy-Induced Lipodystrophy: Atazanavir, a Protease Inhibitor, Compromises Adipogenic Conversion of Adipose-Derived Stem/Progenitor Cells through Accelerating ER Stress-Mediated Cell Death in Differentiating Adipocytes.

Lipodystrophy is a common complication in human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART) or antiretroviral therapy (ART). Previous studies demonstrated that endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) is involved in lipodystrophy; however, the detailed mechanism has not been fully described in human adipogenic cell lineage. We utilized adipose tissue-derived stem cells (ADSCs) obtained from human subcutaneous adipose tissue, and atazanavir (ATV), a protease inhibitor (PI), was administered to ADSCs and ADSCs undergoing adipogenic conversion. Marked repression of adipogenic differentiation was observed when ATV was administered during 10 days of ADSC culture in adipogenic differentiation medium. Although ATV had no effect on ADSCs, it significantly induced apoptosis in differentiating adipocytes. ATV treatment also caused the punctate appearance of CCAAT-enhancer-binding (C/EBP) protein homologous protein (CHOP), and altered expression of CHOP and GRP78/Bip, which are the representation of ER stress, only in differentiating adipocytes. Administration of UPR inhibitors restored adipogenic differentiation, indicating that ER stress-mediated UPR was induced in differentiating adipocytes in the presence of ATV. We also observed autophagy, which was potentiated in differentiating adipocytes by ATV treatment. Thus, adipogenic cell atrophy leads to ATV-induced lipodystrophy, which is mediated by ER stress-mediated UPR and accelerated autophagy, both of which would cause adipogenic apoptosis. As our study demonstrated for the first time that ADSCs are unsusceptible to ATV and its deleterious effects are limited to the differentiating adipocytes, responsible target(s) for ATV-induced lipodystrophy may be protease(s) processing adipogenesis-specific protein(s).