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Beiging of white adipose tissue (WAT) has beneficial effects on metabolism. Although it is known that beige adipocytes are active in lipid catabolism and thermogenesis, how they are regulated deserves more explorations. In this study, we demonstrate that stearoyl-CoA desaturase 1 (SCD1) in subcutaneous WAT (scWAT) responded to cold stimulation and was able to promote mobilization of triacylglycerol [TAG (triglyceride)]. In vitro studies showed that SCD1 promoted lipolysis in C3H10T1/2 white adipocytes. The lipolytic effect was contributed by one of SCD1's products, oleic acid (OA). OA upregulated adipose TAG lipase and hormone-sensitive lipase expression. When SCD1 was overexpressed in the scWAT of mice, lipolysis was enhanced, and oxygen consumption and heat generation were increased. These effects were also demonstrated by the SCD1 knockdown experiments in mice. In conclusion, our study suggests that SCD1, known as an enzyme for lipid synthesis, plays a role in upregulating lipid mobilization through its desaturation product, OA.

Autophagy is a stress-induced lysosomal degradation pathway regulated by evolutionarily conserved autophagy-related (ATG) genes. Recent research has revealed that autophagy plays an important role in the regulation of energy metabolism, development of metabolic tissues, and pathogenesis of metabolic disorders. Bulk and selective degradation by autophagy helps maintain protein homeostasis and physiological function of cells. Aside from classical degradative roles, ATG proteins also carry out non-classical secretory functions of metabolic tissues. In this review, we summarize recent progresses and unanswered questions on the mechanisms of autophagy and ATG proteins in metabolic regulation, with a focus on organelle and nutrient storage degradation, as well as vesicular and hormonal secretion. Such knowledge broadens our understanding on the cause, pathophysiology, and prevention of metabolic diseases including obesity and diabetes.

Increasing attention is now being paid to the important role played by autophagic flux in maintaining normal blood vessel walls. Endothelial cell dysfunction initiates the development of atherosclerosis. In the endothelium, a variety of critical triggers ranging from shear stress to circulating blood lipids promote autophagy. Furthermore, emerging evidence links autophagy to a range of important physiological functions such as redox homeostasis, lipid metabolism, and the secretion of vasomodulatory substances that determine the life and death of endothelial cells. Thus, the promotion of autophagy in endothelial cells may have the potential for treating atherosclerosis. This paper reviews the role of endothelial cells in the pathogenesis of atherosclerosis and explores the molecular mechanisms involved in atherosclerosis development.

Adaptation to changes in energy availability is pivotal for the survival of animals. Adipose tissue, the body's largest reservoir of energy and a major source of metabolic fuel, exerts a buffering function for fluctuations in nutrient availability. This functional plasticity ranges from energy storage in the form of triglycerides during periods of excess energy intake to energy mobilization via lipolysis in the form of free fatty acids for other organs during states of energy demands. The subtle balance between energy storage and mobilization is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance, type 2 diabetes and cancer cachexia. As a result, adipocyte lipolysis is tightly regulated by complex regulatory mechanisms involving lipases and hormonal and biochemical signals that have opposing effects. In thermogenic brown and brite adipocytes, lipolysis stimulation is the canonical way for the activation of non-shivering thermogenesis. Lipolysis proceeds in an orderly and delicately regulated manner, with stimulation through cell-surface receptors via neurotransmitters, hormones, and autocrine/paracrine factors that activate various intracellular signal transduction pathways and increase kinase activity. The subsequent phosphorylation of perilipins, lipases, and cofactors initiates the translocation of key lipases from the cytoplasm to lipid droplets and enables protein-protein interactions to assemble the lipolytic machinery on the scaffolding perilipins at the surface of lipid droplets. Although activation of lipolysis has been well studied, the feedback fine-tuning is less well appreciated. This review focuses on the molecular brakes of lipolysis and discusses some of the divergent fine-tuning strategies in the negative feedback regulation of lipolysis, including delicate negative feedback loops, intermediary lipid metabolites-mediated allosteric regulation and dynamic protein–protein interactions. As aberrant adipocyte lipolysis is involved in various metabolic diseases and releasing the brakes on lipolysis in thermogenic adipocytes may activate thermogenesis, targeting adipocyte lipolysis is thus of therapeutic interest. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb)-driven lipid accumulation is intricately associated with the progression of tuberculosis (TB) disease. Although several studies elucidating the mechanisms for lipid droplet (LD) biosynthesis exist, we provide evidence for the significance of their regulated turnover via macroautophagy/autophagy during Mtb infection. We demonstrate that Mtb utilizes EGFR (epidermal growth factor receptor) signaling to induce the expression of the histone acetylation reader, BRD4 (bromodomain containing 4). The EGFR-BRD4 axis suppresses lipid-specific autophagy, and hence favors cellular lipid accumulation. Specifically, we found that pharmacological inhibition or knockdown of Egfr or Brd4 enhances autophagic flux and concomitantly decreases cellular LDs that is otherwise maintained at a significant level in chloroquine-treated or Atg5 knocked down autophagy-compromised host cells. In line with the enhanced lipophagy, we found that loss of EGFR or BRD4 function restricts mycobacterial burden that is rescued by external replenishment with oleic acid. We also report that the EGFR-BRD4 axis exerts additional effects by modulating pro-angiogenic gene expression and consequently aberrant angiogenesis during mycobacterial infection. This is important in the context of systemic Mtb dissemination as well as for the efficient delivery of anti-mycobacterial therapeutics to the Mtb-rich core of TB granuloma. Finally, utilizing an in vivo mouse model of TB, we show that pharmacological inhibition of EGFR and BRD4 compromises LD buildup via enhanced lipophagy and normalizes angiogenesis, thereby restricting Mtb burden and rescuing mice from severe TB-like pathology. These findings shed light on the novel roles of BRD4 during Mtb infection, and its possible implication in potentiating anti-TB responses. Abbreviations: ATG5: autophagy related 5; BRDs: bromodomain containing; COL18A1: collagen type XVIII alpha 1 chain; EGFR: epidermal growth factor receptor; EP300: E1A binding protein p300; KDR: kinase insert domain receptor; KLF5: Kruppel like factor 5; LDs: lipid droplets; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; Mtb: Mycobacterium tuberculosis; PECAM1: platelet and endothelial cell adhesion molecule 1; SQSTM1/p62: sequestosome 1; TB: tuberculosis; THBS1: thrombospondin 1; VEGF: vascular endothelial growth factor [ABSTRACT FROM AUTHOR]

Insulin resistance (IR) attributed by the deficiency of lipophagy, is an abnormal state of downregulation of insulin-mediated glucose uptake and use into the liver. Chromosome 9 open reading frame 72 (C9orf72) variously modulates autophagy. We investigated the role and the downstream pathway of C9orf72 in hepatic IR. We found that C9orf72 knockdown alleviated hepatic IR by lipophagy promotion in T2DM mice and in IR-challenged hepatocytes in vitro. C9orf72 interacted with and activated cell division cycle 42 (Cdc42) protein in IR-challenged hepatocytes, Which in turn, inhibits lipophagy by promoting neural Wiskott-Aldrich syndrome protein (N-WASP) expression and activation. C9orf72 inhibited lipophagy by activating the Cdc42/N-WASP axis to facilitate hepatic IR; therefore, the knockdown of C9orf72 may be potentially therapeutic for the treatment of IR. • Knockdown of C9orf72 alleviated hepatic insulin resistance via facilitating lipophagy in T2DM mice and in insulin resistance-challenged hepatocytes in vitro. • C9orf72 interacted with and activated Cdc42 in insulin resistance-challenged hepatocytes. • The activation of Cdc42 inhibited lipophagy in insulin resistance-challenged hepatocytes via enhancing N-WASP expression and activation. [ABSTRACT FROM AUTHOR]

Alcohol's impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol's inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD.

Nutrient starvation initiates cell cycle exit and entry into quiescence, a reversible, non-proliferative state characterized by stress tolerance, longevity and large-scale remodeling of subcellular structures. Depending on the nature of the depleted nutrient, yeast cells are assumed to enter heterogeneous quiescent states with unique but mostly unexplored characteristics. Here, we show that storage and consumption of neutral lipids in lipid droplets (LDs) differentially impacts the regulation of quiescence driven by glucose or phosphate starvation. Upon prolonged glucose exhaustion, LDs were degraded in the vacuole via Atg1-dependent lipophagy. In contrast, yeast cells entering quiescence due to phosphate exhaustion massively over-accumulated LDs that clustered at the vacuolar surface but were not engulfed via lipophagy. Excessive LD biogenesis required contact formation between the endoplasmic reticulum and the vacuole at nucleus-vacuole junctions and was accompanied by a shift of the cellular lipid profile from membrane towards storage lipids, driven by a transcriptional upregulation of enzymes generating neutral lipids, in particular sterol esters. Importantly, sterol ester biogenesis was critical for long-term survival of phosphate-exhausted cells and supported rapid quiescence exit upon nutrient replenishment, but was dispensable for survival and regrowth of glucose-exhausted cells. Instead, these cells relied on de novo synthesis of sterols and fatty acids for quiescence exit and regrowth. Phosphate-exhausted cells efficiently mobilized storage lipids to support several rounds of cell division even in presence of inhibitors of fatty acid and sterol biosynthesis. In sum, our results show that neutral lipid biosynthesis and mobilization to support quiescence maintenance and exit is tailored to the respective nutrient scarcity.

Objective: Diet-induced obesity is often associated with nonalcoholic fatty liver disease (NAFLD), which instigates severe metabolic disorders, including cirrhosis, hepatocellular carcinoma, and type 2 diabetes. We have shown that hepatic depletion of CREB regulated transcription co-activator (CRTC) 2 protects mice from the progression of diet-induced fatty liver phenotype, although the exact mechanism by which CRTC2 modulates this process is elusive to date. Here, we investigated the role of hepatic CRTC2 in the instigation of NAFLD in mammals. Methods: Crtc2 liver-specific knockout (Crtc2 LKO) mice and Crtc2 flox/flox (Crtc2 f/f) mice were fed a high fat diet (HFD) for 7–8 weeks. Body weight, liver weight, hepatic lipid contents, and plasma triacylglycerol (TG) levels were determined. Western blot analysis was performed to determine Sirtuin (SIRT) 1, tuberous sclerosis complex (TSC) 2, and mammalian target of rapamycin complex (mTORC) 1 activity in the liver. Effects of Crtc2 depletion on lipogenesis was determined by measuring lipogenic gene expression (western blot analysis and qRT-PCR) in the liver as well as Oil red O staining in hepatocytes. Effects of miR-34a on mTORC1 activity and hepatic lipid accumulation was assessed by AAV-miR-34a virus in mice and Ad-miR-34a virus and Ad-anti-miR-34a virus in hepatocytes. Autophagic flux was assessed by western blot analysis after leupeptin injection in mice and bafilomycin treatment in hepatocytes. Lipophagy was assessed by transmission electron microscopy and confocal microscopy. Expression of CRTC2 and p-S6K1 in livers of human NAFLD patients was assessed by immunohistochemistry. Results: We found that expression of CRTC2 in the liver is highly induced upon HFD-feeding in mice. Hepatic depletion of Crtc2 ameliorated HFD-induced fatty liver disease phenotypes, with a pronounced inhibition of the mTORC1 pathway in the liver. Mechanistically, we found that expression of TSC2, a potent mTORC1 inhibitor, was enhanced in Crtc2 LKO mice due to the decreased expression of miR-34a and the subsequent increase in SIRT1-mediated deacetylation processes. We showed that ectopic expression of miR-34a led to the induction of mTORC1 pathway, leading to the hepatic lipid accumulation in part by limiting lipophagy and enhanced lipogenesis. Finally, we found a strong association of CRTC2, miR-34a and mTORC1 activity in the NAFLD patients in humans, demonstrating a conservation of signaling pathways among species. Conclusions: These data collectively suggest that diet-induced activation of CRTC2 instigates the progression of NAFLD by activating miR-34a-mediated lipid accumulation in the liver via the simultaneous induction of lipogenesis and inhibition of lipid catabolism. Therapeutic approach to specifically inhibit CRTC2 activity in the liver could be beneficial in combating NAFLD in the future.

The liver is the most significant metabolic organ in the body and plays an important role in lipid metabolism. Liver lipid metabolism disorders cause hepatic diseases such as hepatitis, hepatic cirrhosis, and hepatoma. Autophagy is a process of generating energy and building blocks by degrading redundant or damaged proteins and organelles. Thus, it helps in the maintenance of cellular homeostasis. Recent discoveries revealed that lipophagy plays a vital role in hepatic cellular homeostasis and lipid metabolism. Its imbalance is always associated with the perturbation of lipid metabolism in the liver. This article reviewed the molecular mechanisms involved in lipophagy and the interaction between lipophagy and hepatic lipid metabolism. Increasing evidence suggests that lipophagy is an effective method to resolve liver diseases.

Porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus genus within the Coronavirus family, causing severe watery diarrhea in piglets and resulting in significant economic losses. Medium-chain acyl-CoA dehydrogenase (ACADM) is an enzyme participating in lipid metabolism associated with metabolic diseases and pathogen infections. Nonetheless, the precise role of ACADM in regulating PEDV replication remains uncertain. In this study, we identified ACADM as the host binding partner of NSP4 via immunoprecipitation-mass spectrometry analysis. The interaction between ACADM and NSP4 was subsequently corroborated through coimmunoprecipitation and laser confocal microscopy. Following this, a notable upsurge in ACADM expression was observed during PEDV infection. ACADM overexpression effectively inhibited virus replication, whereas ACADM knockdown facilitated virus replication, suggesting ACADM has negative regulation effect on PEDV infection. Furthermore, we demonstrated fatty acid β-oxidation affected PEDV replication for the first time, inhibition of fatty acid β-oxidation reduced PEDV replication. ACADM decreased PEDV-induced β-oxidation to suppress PEDV replication. Mechanistically, ACADM reduced cellular free fatty acid levels and subsequent β-oxidation by hindering AMPK-mediated lipophagy. In summary, our results reveal that ACADM plays a negative regulatory role in PEDV replication by regulating lipid metabolism. The present study introduces a novel approach for the prevention and control of PEDV infection., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ovarian clear cell carcinoma (OCCC) is a histological subtype that constitutes approximately 20% of epithelial ovarian cancer cases in Asian countries, but has a relatively low incidence in Western countries. Meanwhile, clear cell renal cell carcinoma (ccRCC) is a major subtype of kidney cancer. OCCC and ccRCC resemble one another histologically and have clear cytoplasmic appearances. Studies have revealed some genetic similarities between OCCC and ccRCC. However, information regarding common biological background factors between these cancers remains scarce. For example, accumulation of cellular lipid droplets was shown to play a crucial role in ccRCC progression, while similar information is lacking for OCCC. In this perspective article, we propose that lipid droplets may be candidates for future exploration to better understand the common biological backgrounds between OCCC and ccRCC, potentially leading to subtype-specific treatment strategies. We further discuss the relationship between poly ADP-ribose polymerase inhibition treatment and lipid metabolism because this therapeutic strategy has attracted considerable attention as a treatment for epithelial ovarian cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Koizume, Takahashi and Miyagi.)

Hexagonal boron nitride (hBN) is an emerging 2D material attracting significant attention due to its superior electrical, chemical, and therapeutic properties. However, inhalation toxicity mechanisms of hBN in human lung cells are poorly understood. Here, cellular interaction and effects of hBN nanosheets is investigated in alveolar epithelial cells cultured on porous inserts and exposed under air-liquid interface conditions for 24 h. hBN is taken up by the cells as determined in a label-free manner via RAMAN-confocal microscopy, ICP-MS, TEM, and SEM-EDX. No significant (p > 0.05) effects are observed on cell membrane integrity (LDH release), epithelial barrier integrity (TEER), interleukin-8 cytokine production or reactive oxygen production at tested dose ranges (1, 5, and 10 µg cm -2 ). However, it is observed that an enhanced accumulation of lipid granules in cells indicating the effect of hBN on lipid metabolism. In addition, it is observed that a significant (p < 0.05) and dose-dependent (5 and 10 µg cm -2 ) induction of autophagy in cells after exposure to hBN, potentially associated with the downstream processing and breakdown of excess lipid granules to maintain lipid homeostasis. Indeed, lysosomal co-localization of lipid granules supporting this argument is observed. Overall, the results suggest that the continuous presence of excess intracellular lipids may provoke adverse outcomes in the lungs., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Background/aims: Metabolic dysfunction-associated steatohepatitis (MASH) is an unmet clinical challenge due to the rapid increased occurrence but lacking approved drugs. Autophagy-related protein 16-like 1 (ATG16L1) plays an important role in the process of autophagy, which is indispensable for proper biogenesis of the autophagosome, but its role in modulating macrophage-related inflammation and metabolism during MASH has not been documented. Here, we aimed to elucidate the role of ATG16L1 in the progression of MASH., Methods: Expression analysis was performed with liver samples from human and mice. MASH models were induced in myeloid-specific Atg16l1-deficient and myeloid-specific Atg16l1-overexpressed mice by high-fat and high-cholesterol diet or methionine- and choline-deficient diet to explore the function and mechanism of macrophage ATG16L1 in MASH., Results: Macrophage-specific Atg16l1 knockout exacerbated MASH and inhibited energy expenditure, whereas macrophage-specific Atg16l1 transgenic overexpression attenuated MASH and promotes energy expenditure. Mechanistically, Atg16l1 knockout inhibited macrophage lipophagy, thereby suppressing macrophage β-oxidation and decreasing the production of 4-hydroxynonenal, which further inhibited stimulator of interferon genes(STING) carbonylation. STING palmitoylation was enhanced, STING trafficking from the endoplasmic reticulum to the Golgi was promoted, and downstream STING signaling was activated, promoting proinflammatory and profibrotic cytokines secretion, resulting in hepatic steatosis and hepatic stellate cells activation. Moreover, Atg16l1-deficiency enhanced macrophage phagosome ability but inhibited lysosome formation, engulfing mtDNA released by pyroptotic hepatocytes. Increased mtDNA promoted cGAS/STING signaling activation. Moreover, pharmacological promotion of ATG16L1 substantially blocked MASH progression., Conclusion: ATG16L1 suppresses MASH progression by maintaining macrophage lipophagy, restraining liver inflammation, and may be a promising therapeutic target for MASH management.

Lipophagy, a type of autophagy that breaks down lipid droplets, is essential in the regulation of intracellular lipid accumulation and intracellular free fatty acid levels in numerous organisms and metabolic conditions. We investigated the effects of Stevia rebaudiana Bertoni (S), a low-calorie sweetener, and stevioside (SS) on hepatic steatosis and autophagy in hepatocytes, as well as in db/db mice. S and SS reduced the body and liver weight and levels of serum triglyceride, total cholesterol, and hepatic lipogenic proteins. In addition, S and SS increased the levels of fatty acid oxidase, peroxisome proliferator-activated receptor alpha (PPARα), and microtubule-associated protein light chain 3 B but decreased that of sequestosome 1 (p62) in the liver of db/db mice. Additionally, Beclin 1, lysosomal associated membrane protein 1, and phosphorylated adenosine monophosphate-activated protein kinase protein expression was augmented following S and SS treatment of db/db mice. Furthermore, the knockdown of PPARα blocked lipophagy in response to SS treatment in HepG2 cells. These outcomes indicate that PPARα-dependent lipophagy is involved in hepatic steatosis in the db/db mouse model and that SS, a PPARα agonist, represents a new therapeutic option for managing associated diseases.

The global prevalence of nonalcoholic fatty liver disease (NAFLD) defined by the increased number of lipid droplets (LDs) in hepatocytes, have risen continuously in parallel with the obesity. LDs and related proteins are known to affect cellular metabolism and signaling. Seipin, one of the most important LD-related proteins, plays a critical role in LD biogenesis. Although the role of adipose tissue-specific Seipin silencing is known, hepatocyte-specific silencing upon cholesterol-mediated lipid accumulation has not been investigated. In our study, we investigated the effect of Seipin on endoplasmic reticulum (ER) stress and lipophagy in cholesterol accumulated mouse hepatocyte cells. In this direction, cholesterol accumulation was induced by cholesterol-containing liposome, while Seipin mRNA and protein levels were reduced by siRNA. Our findings show that cholesterol containing liposome administration in hepatocytes increases both Seipin protein and number of large LDs. However Seipin silencing reduced the increase of cholesterol mediated large LDs and Glucose-regulated protein 78 (GRP78) mRNA. Additionally, lysosome-LD colocalization increased only in cells treated with cholesterol containing liposome, while the siRNA against Seipin did not lead any significant difference. According to our findings, we hypothesize that Seipin silencing in hepatocytes reduced cholesterol mediated LD maturation as well as GRP78 levels, but not lipophagy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Macroautophagy/autophagy requires enormous membrane expansions during concerted actions of transient autophagic vesicles and lysosomes, yet the source of the membrane lipids is poorly understood. Recent work in adipocytes has now pinpointed the de novo lipogenesis pathway as the preferred source of fatty acids for phospholipid in autophagic membrane synthesis, as loss of FASN (fatty acid synthase) disrupts autophagic flux and lysosome function in vivo and in vitro. These data indicate fatty acid synthesis channels lipid for membrane expansions, whereas fatty acids from circulating lipoproteins provide for adipose lipid storage. Importantly, autophagy blockade upon loss of fatty acids promotes a strong thermogenic phenotype in adipocytes, another striking example whereby autophagy controls cell behavior. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is a global health problem that develops through unclear molecular mechanisms. The P2X7 purinergic receptor (P2RX7) is an ATP‐gated ion channel that belongs to the P2XR family. Thus far, studies on P2RX7 in NAFLD have been largely contradictory. Integrating experiments and modeling, we elucidate the dynamic processes of lipid droplet fusion and degradation following regulation of P2RX7. We show that activation of P2RX7 can activate the AMPK/ULK1 pathway to promote autophagosome generation and lysosomal degradation of autophagosomes. Inhibiting P2RX7 has the opposite effect. Notably, we find that lipid droplets become larger by the fusion of dysfunctional lysosomes but cannot be degraded by them following P2RX7 inhibition. Our study provides evidence that P2RX7 activation improves NAFLD by promoting lipophagy. [ABSTRACT FROM AUTHOR]

Background: Non-alcoholic fatty liver disease (NAFLD) is characterized by ectopic accumulation of triglycerides in the liver. Emerging evidence has demonstrated that lipophagy regulates lipid mobilization and energy homeostasis in the liver. Sirtuin 3 (SIRT3), a mitochondrial NAD+-dependent deacetylase, modulates the activities of several substrates involving in autophagy and energy metabolism. Honokiol (HK) is a natural lignan from the plants of Magnolia genus that exhibits potent liver protective property. Methods: AML12 was challenged with 500 μM palmitic acid and 250 μM oleic acid mixture solution to induce lipotoxicity. C57BL/6J mice were fed with a choline-deficient high fat diet (CDHFD) to generate liver steatosis. The expression of autophagy-related and AMP-activated protein kinase (AMPK) pathway proteins was evaluated by Western blotting and immunofluorescence staining. Intracellular lipid accumulation was validated by Nile red staining. Molecular docking analysis was performed on AutoDock 4.2. Results: HK (5 and 10 μM) was found to attenuate lipid accumulation through promoting SIRT3-AMPK-mediated autophagy, mainly on lipid droplets. HK had hydrophobic interaction with amino acid residues (PHE294, GLU323 and VAL324) and NAD+. Moreover, HK improved mitochondrial function to enhance lipolysis, through decreasing the acetylated long-chain acyl-CoA dehydrogenase level. In CDHFD-fed mice, HK (2.5 and 10 mg/Kg) treatment obviously prevented lipid accumulation in the liver. And co-treatment of the AMPK inhibitor, Compound C, almost abolished the above changes. Conclusions: These results suggest that HK could ameliorate lipotoxicity in hepatocytes by activating SIRT3-AMPK-lipophagy axis, which might be a potential therapeutic agent against NAFLD. [ABSTRACT FROM AUTHOR]

Background: Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein inducing dimerization with cytochrome P450, which mediates chemoresistance. Increased PGRMC1 expression is found in multiple types of resistant cancers, but the role of PGRMC1 in the ferroptosis of cancer cells remains unrevealed. Therefore, we examined the role of PGRMC1 in promoting ferroptosis in paclitaxel-tolerant persister cancer cells (PCC). Methods: The effects of ferroptosis inducers and PGRMC1 gene silencing/overexpression were tested on head and neck cancer (HNC) cell lines and mouse tumor xenograft models. The results were analyzed about cell viability, death, lipid ROS and iron production, mRNA/protein expression and interaction, and lipid assays. Results: PCC had more free fatty acids, lipid droplets, and fatty acid oxidation (FAO) than their parental cells. PCC was highly sensitive to inhibitors of system xc− cystine/glutamate antiporter (xCT), such as erastin, sulfasalazine, and cyst(e)ine deprivation, but less sensitive to (1S,3R)-RSL3. PGRMC1 silencing in PCC reduced ferroptosis sensitivity by xCT inhibitors, and PGRMC1 overexpression in parental cells increased ferroptosis by xCT inhibitors. Lipid droplets were degraded along with autophagy induction and autophagosome formation by erastin treatment in PCC. Lipophagy was accompanied by increased tubulin detyrosination, which was increased by SIRT1 activation but decreased by SIRT1 inhibition. FAO and lipophagy were also promoted by the interaction between lipid droplets and mitochondria. Conclusion: PGRMC1 expression increased FAO and ferroptosis sensitivity from in vivo mice experiments. Our data suggest that PGRMC1 promotes ferroptosis by xCT inhibition in PCC. Paclitaxel-tolerant persister cancer cells (PCC) had PGRMC1 upregulation related to increased free fatty acids, lipid droplets, and fatty acid oxidation. PGRMC1 expression substantially increased ferroptosis by xCT inhibition via lipophagy and tubulin detyrosination, whereas PGRMC1 silencing decreased ferroptosis: this suggests that PGRMC1 expression promotes ferroptosis in PCC. [ABSTRACT FROM AUTHOR]

Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, "lipophagy" refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5, and Scarb2, significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis. Abbreviations: ADGRL3: adhesion G protein-coupled receptor L3; agLDL: aggregated low density lipoprotein; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BMDM: bone-marrow derived macrophages; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; BSA: bovine serum albumin; CALCOCO2: calcium binding and coiled-coil domain 2; CIRBP: cold inducible RNA binding protein; COLGALT1: collagen beta(1-O)galactosyltransferase 1; CORO1A: coronin 1A; DMA: deletion mutant array; Faa4: long chain fatty acyl-CoA synthetase; FBS: fetal bovine serum; FUS: fused in sarcoma; HMGB1: high mobility group box 1; HMGB2: high mobility group box 2: HSP90AA1: heat shock protein 90: alpha (cytosolic): class A member 1; HSPA5: heat shock protein family A (Hsp70) member 5; HSPA8: heat shock protein 8; HSPB1: heat shock protein 1; HSPH1: heat shock 105kDa/110kDa protein 1; LDAH: lipid droplet associated hydrolase; LIPA: lysosomal acid lipase A; LIR: LC3-interacting region; MACROH2A1: macroH2A.1 histone; MAP1LC3: microtubule-associated protein 1 light chain 3; MCOLN1: mucolipin 1; NBR1: NBR1, autophagy cargo receptor; NPC2: NPC intracellular cholesterol transporter 2; OPTN: optineurin; P/S: penicillin-streptomycin; PLIN2: perilipin 2; PLIN3: perilipin 3; PNPLA2: patatin like phospholipase domain containing 2; RAB: RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR: selective autophagy receptor; SCARB2: scavenger receptor class B, member 2; SGA: synthetic genetic array; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB: transcription factor EB; TOLLIP: toll interacting protein; UBE2G2: ubiquitin conjugating enzyme E2 G2; UVRAG: UV radiation resistance associated gene; VDAC2: voltage dependent anion channel 2; VIM: vimentin [ABSTRACT FROM AUTHOR]

Although macroautophagy/autophagy has been proposed as a critical defense mechanism against HIV-1 by targeting viral components for degradation, its contribution as a catabolic process in providing optimal anti-HIV-1 immunity has never been addressed. The failure to restore proper antiviral CD8A/CD8 T-cell immunity, especially against HIV-1, is still the major limitation of current antiretroviral therapies. Consequently, it is of clinical imperative to provide new strategies to enhance the function of HIV-1-specific CD8A T-cells in patients under antiretroviral treatments (ART). Here, we investigated whether targeting autophagy activity could be an optional solution to make this possible. Our data show that, after both polyclonal and HIV-1-specific activation, CD8A T-cells from ART displayed reduced autophagy-dependent degradation of lysosomal contents when compared to naturally HIV-1 protected elite controllers (EC). We further confirmed in EC, by using specific BECN1 gene silencing and lysosomal inhibitors, the critical role of active autophagy in superior CD8A T-cell protection against HIV-1. More importantly, we found that an IL21 treatment was effective in rescuing the antiviral CD8A T-cell immunity from ART in an autophagy-dependent manner. Finally, we established that IL21-dependent rescue occurred due to the enhanced degradation of endogenous lipids via autophagy, referred to as lipophagy, which fueled the cellular rates of mitochondrial beta-oxidation. In summary, our data show that autophagy/lipophagy can be considered as a therapeutic tool to elicit functional antiviral CD8 T-cell responses. Our results also provide additional insights toward the development of improved T-cell-based prevention and cure strategies against HIV-1. Abbreviations: ART: patients under antiretroviral therapy; BaF: bafilomycin A1; BECN1: beclin 1; CEF: cytomegalo-, Epstein-Barr- and flu-virus peptide pool; Chloro.: chloroquine; EC: elite controllers; FAO: fatty acid beta-oxidation; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBMC: peripheral blood mononuclear cells; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1. [ABSTRACT FROM AUTHOR]

Selective autophagy is a specific elimination of certain intracellular substrates by autophagic pathways. The most studied macroautophagy pathway involves tagging and recognition of a specific cargo by the autophagic membrane (phagophore) followed by the complete sequestration of targeted cargo from the cytosol by the double-membrane vesicle, autophagosome. Until recently, the knowledge about selective macroautophagy was minimal, but now there is a panoply of links elucidating how phagophores engulf their substrates selectively. The studies of selective autophagy processes have further stressed the importance of using the in vivo models to validate new in vitro findings and discover the physiologically relevant mechanisms. However, dissecting how the selective autophagy occurs yet remains difficult in living organisms, because most of the organelles are relatively inaccessible to observation and experimental manipulation in mammals. In recent years, zebrafish (Danio rerio) is widely recognized as an excellent model for studying autophagic processes in vivo because of its optical accessibility, genetic manipulability and translational potential. Several selective autophagy pathways, such as mitophagy, xenophagy, lipophagy and aggrephagy, have been investigated using zebrafish and still need to be studied further, while other selective autophagy pathways, such as pexophagy or reticulophagy, could also benefit from the use of the zebrafish model. In this review, we shed light on how zebrafish contributed to our understanding of these selective autophagy processes by providing the in vivo platform to study them at the organismal level and highlighted the versatility of zebrafish model in the selective autophagy field. Abbreviations: AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; Atg: autophagy-related; CMA: chaperone-mediated autophagy; CQ: chloroquine; HsAMBRA1: human AMBRA1; KD: knockdown; KO: knockout; LD: lipid droplet; MMA: methylmalonic acidemia; PD: Parkinson disease; Tg: transgenic. [ABSTRACT FROM AUTHOR]

SUMMARY: Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation conditions and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12 h post‐illumination. We observed that while gene expression for photosynthetic machinery during de‐etiolation was largely unaffected in atg mutants, several genes involved in photosystem assembly were transcriptionally downregulated. We also investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in chloroplast development. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected de‐etiolation. Moreover, we showed that lower levels of carbon resources exacerbated the slow greening phenotype whereas higher levels of carbon resources had an opposite effect. This work suggests a lack of autophagy machinery limits chloroplast development during de‐etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development. Significance Statement: Autophagy is a catabolic process that plays an essential role during different developmental processes. We show that lack of autophagy machinery limits chloroplast development during de‐etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development. [ABSTRACT FROM AUTHOR]

The prostate gland is surrounded by periprostatic adipose tissue (PPAT), which is believed to play a role in prostate cancer (PCa) progression. Cancer cells can take up lipids from the microenvironment and store them in lipid droplets (LDs). Fatty acids released from LDs are used by PCa cells as preferential metabolic fuels to provide energy and promote cancer progression. Recently, fatty acids have been associated with autophagy, a cellular recycling pathway. Lipophagy is a selective form of autophagy involved in LD degradation, the role of which in PCa progression remains unknown. Here, we explored markers of autophagy and lipophagy in human PCa tissues in correlation with factors of aggressiveness, and we evaluated the influence of PPAT adipocytes on autophagy and lipophagy. We analyzed markers of autophagy (p62, LC3), lipid droplets (PLIN and Oil Red O), androgen receptor (AR), proliferation (Ki67), and epithelial–mesenchymal transition (Zeb1) on 465 PCa samples. Co‐cultures of PCa cell lines PC3 and 22RV1 with adipocytes isolated from patients' PPAT were used to analyze the influence of PPAT on autophagy and lipophagy in vitro. In human PCa tissues, we observed a correlation between markers of LD and those of autophagy, which are associated with clinical and biological factors of disease aggressiveness. In addition, PLIN staining was associated with AR expression. In locally advanced PCa, p62, LC3, and PLIN were increased in extraprostatic areas where cancer cells are in contact with PPAT. Co‐culture of PCa cell lines with adipocytes decreased autophagy activity and increased LD flux in PC3 cells. These results suggest an active process of lipophagy in PCa, linked to disease aggressiveness, to the proximity of PPAT, and induced in vitro in co‐culture with adipocytes. Lipophagy is therefore likely to be a crucial player in PCa progression. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Recent evidence demonstrated that chronic intake of quercetin attenuated hepatic fat accumulation in various animal models of obesity and diabetes. However, whether quercetin has the ability to enhance energy metabolism in hepatocytes and its exact mechanisms have yet to be identified. In the present study, we investigated whether quercetin directly enhanced the energy metabolism of cultured hepatocytes by focusing on lipophagy, involving selective autophagic degradation of lipid droplets. As an indicator of mitochondrial respiration, oxygen consumption was measured following 12-h treatment with quercetin or its related flavonoids, isorhamnetin and rutin (10 μM) using an extracellular flux analyzer. Treatment of alpha mouse liver 12 (AML12) hepatocytes with quercetin enhanced mitochondrial respiration, but isorhamnetin and rutin did not. Results of a palmitate-bovine serum albumin fatty acid oxidation assay showed that quercetin significantly increased the oxygen consumption of AML12 hepatocytes, suggesting enhanced fatty acid β-oxidation. However, as expression levels of mitochondrial oxidative phosphorylation proteins were unaltered by quercetin, we explored whether lipophagy contributed to enhanced fatty acid β-oxidation. Increased colocalization of lipid droplets and lysosomes confirmed that quercetin promoted lipophagy in AML12 hepatocytes. Furthermore, pharmacological inhibition of the autophagy–lysosomal pathway abolished the enhancement of fatty acid β-oxidation induced by quercetin in AML12 hepatocytes, suggesting that the enhancement of lipophagy by quercetin contributed to increased fatty acid β-oxidation. Finally, we showed that quercetin could activate AMPK signaling, which regulates autophagy even under nutrient-sufficient conditions. Our findings indicate that quercetin enhanced energy metabolism by a potentially novel mechanism involving promotion of lipophagy to produce the substrate for fatty acid β-oxidation in mitochondria through activation of AMPK signaling. Our results suggest the possibility that nutrient-induced lipophagy might contributes to the reduction of fat in hepatocytes.

Burkholderia pseudomallei: which causes melioidosis with high mortality in humans, has become a global public health concern. Recently, infection-driven lipid droplet accumulation has been related to the progression of host-pathogen interactions, and its contribution to the pathogenesis of infectious disease has been investigated. Here, we demonstrated that B. pseudomallei infection actively induced a time-dependent increase in the number and size of lipid droplets in human lung epithelial cells and macrophages. We also found that lipid droplet accumulation following B. pseudomallei infection was associated with downregulation of PNPLA2/ATGL (patatin like phospholipase domain containing 2) and lipophagy inhibition. Functionally, lipid droplet accumulation, facilitated via PNPLA2 downregulation, inhibited macroautophagic/autophagic flux and, thus, hindered autophagy-dependent inhibition of B. pseudomallei infection in lung epithelial cells. Mechanistically, we further revealed that nuclear receptor NR1D2 might be involved in the suppression of PNPLA2 after cell exposure to B. pseudomallei. Taken together, our findings unraveled an evolutionary strategy, by which B. pseudomallei interferes with the host lipid metabolism, to block autophagy-dependent suppression of infection. This study proposes potential targets for clinical therapy of melioidosis. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG7: autophagy related 7; B. pseudomallei: Burkholderia pseudomallei; CFU: colony-forming unit; DG: diglyceride; FASN: fatty acid synthase; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LD: lipid droplet; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MG: monoglyceride; MOI: multiplicity of infection; mRFP: monomeric red fluorescent protein; NR1D2: nuclear receptor subfamily 1 group D member 2; p.i., post-infection; PLIN2/ADRP: perilipin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; shRNA: short hairpin RNA; TEM: transmission electron microscopy; TG: triglyceride [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved process for degrading intracellular materials in vacuoles or lysosomes, commonly found in a wide range of eukaryotes. Lipid droplets are intracellular organelles that store neutral lipids, and recent studies have shown that autophagy regulates lipid metabolism by degrading lipid droplets. While autophagy-mediated degradation of lipid droplets, called lipophagy, is recognized as one of the major lipid degradation mechanisms in eukaryotic cells, its characterization in microalgae remains incomplete. Our prior investigations established a potential connection between autophagy and lipid degradation on the oleaginous microalga (diatom) Fistulifera solaris , a candidate for microalgal biofuel production. Inhibition of lipophagy can be a promising strategy to enhance the oleaginous phenotype of this alga. However, the presence of lipophagy in this alga has not been demonstrated because the interaction between vacuoles and lipid droplets has not yet been observed. In this study, we investigated lipophagy in F. solaris by microscopy, transcriptomics, and biochemical assays. Fluorescence microscopy revealed increased vacuolar contact with lipid droplets under conditions promoting lipid degradation, and this process was hindered by autophagy inhibitors. Transmission electron microscopy showed direct fusion of vacuoles and lipid droplets, suggesting that F. solaris might have a novel type of microlipophagy. These data indicate that lipophagy may play an important role in lipid degradation in F. solaris. To our knowledge this study marks the first direct observation of lipophagy in diatoms, contributing to our understanding of lipid metabolism in this organism. • Lipophagy analysis in the oleaginous diatom. • Lipophagy have an important role in lipid degradation. • Interaction between vacuoles and lipid droplet promotes lipophagy. [ABSTRACT FROM AUTHOR]

The lipid droplet (LD) organization proteins Ldo16 and Ldo45 affect multiple aspects of LD biology in yeast. They are linked to the LD biogenesis machinery seipin, and their loss causes defects in LD positioning, protein targeting, and breakdown. However, their molecular roles remained enigmatic. Here, we report that Ldo16/45 form a tether complex with Vac8 to create vacuole lipid droplet (vCLIP) contact sites, which can form in the absence of seipin. The phosphatidylinositol transfer protein (PITP) Pdr16 is a further vCLIP-resident recruited specifically by Ldo45. While only an LD subpopulation is engaged in vCLIPs at glucose-replete conditions, nutrient deprivation results in vCLIP expansion, and vCLIP defects impair lipophagy upon prolonged starvation. In summary, Ldo16/45 are multifunctional proteins that control the formation of a metabolically regulated contact site. Our studies suggest a link between LD biogenesis and breakdown and contribute to a deeper understanding of how lipid homeostasis is maintained during metabolic challenges. [Display omitted] • Lipid droplets (LDs) and vacuoles form metabolically controlled vCLIP contact sites • vCLIP comprises the tethers Ldo16/45, the vacuolar adaptor Vac8, and the PITP Pdr16 • The Ldo16/45 partner seipin is dispensable for formation of vCLIP contact sites • vCLIP-based LD-vacuole tethering is required for selective LD autophagy (lipophagy) Diep et al. identify the molecular machinery of the yeast vacuole lipid droplet contact site vCLIP, which comprises two Vac8-dependent tethers, Ldo16 and Ldo45, and the phosphatidylinositol transfer protein Pdr16. vCLIP abundance and molecular composition are metabolically controlled, and vCLIP-dependent vacuole tethering is required for selective lipid droplet autophagy (lipophagy). [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy. [Display omitted] • LDO proteins bind Vac8 to form vCLIP, the vacuole-lipid droplet contact site • Nutrient exhaustion drives vCLIP formation to promote lipophagy • vCLIP is critical for lipophagy and supports caloric-restriction-induced longevity • Disruption of vCLIP induces NVJ expansion and microautophagy of the nucleus Álvarez-Guerra et al. identify the tethering machinery of the membrane contact site that enables lipid droplet consumption via lipophagy. They show that nutrient exhaustion drives the formation of vCLIP, the vacuole-lipid droplet contact site established by the LDO proteins that bind vacuolar Vac8. [ABSTRACT FROM AUTHOR]

Excessive hepatic lipid droplets (LDs) accumulation-induced lipid metabolism disorder contributes to the development of non-alcoholic fatty liver disease (NAFLD). Exercise is a promising therapeutic strategy for NAFLD. However, the mechanism by which exercise ameliorates NAFLD through regulating the catabolism of hepatic LDs remains unclear. In the present study, we investigated the effect of perilipin2 (PLIN2)–lysosomal acid lipase (LIPA) axis mediating exercise-triggered lipophagy in a high-fat diet (HFD)-induced NAFLD mouse model. Our results showed that exercise could reduce HFD-induced hepatic LDs accumulation and change the expression of lipolysis-related enzymes. Moreover, exercise upregulated the expression of microtubule associated protein 1 light chain 3 (LC3) and autophagy-related proteins, and downregulated sequestosome 1 (P62) expression and promoted autophagosomes formation. Interestingly, exercise downregulated PLIN2 expression, upregulated LIPA expression, and increased the activity of hepatic LIPA and serum levels of LIPA in the NAFLD mouse model. Further mechanistic studies demonstrated that adenosine monophosphate-activated protein kinase (AMPK) activator-5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) treatment significantly increased mRNA levels and protein expression of LIPA and LC3II and decreased levels of PLIN2 and P62 in palmitic acid (PA)-treated HepG2 cells. PLIN2 silencing and LIPA overexpression notably increased the mRNA level and protein expression of LC3II and decreased the mRNA level and protein expression of p62, respectively. In summary, our findings reveal novel insights into the effect of exercise on improving lipid droplet metabolism disorder in NAFLD. Enhancing the PLIN2–LIPA axis-mediated lipophagy may be one of the key mechanisms involved in NAFLD alleviation by exercise. • Exercise enhances lipid droplets catabolism by promoting AMPKα-dependent lipophagy. • Exercise regulates lipophagy via the PLIN2-LIPA axis. • Exercise alleviates NAFLD through promoting the PLIN2-LIPA axis-mediated AMPKα-dependent lipophagy. [ABSTRACT FROM AUTHOR]

Lipophagy is a vital biological process that maintains the balance of intracellular lipid metabolism in nonalcoholic fatty liver disease (NAFLD). However, the precise regulatory mechanism of RNF186 in hepatic lipophagy is still unclear. This study investigates the roles and mechanisms of RNF186 in the regulation of lipophagy during the development of NAFLD. In this study, we employed RNF186 knockout mice as well as human liver cells and mouse primary hepatocytes (MPHs) to investigate the role and mechanisms of RNF186 in lipophagy during the progression of NAFLD. Additionally, liver specimens from individuals with NAFLD were examined to assess the expression of RNF186 and its associated factors. Here, we provide evidence that depletion of RNF186 enhances lipophagy in hepatocytes of a NAFLD model. Mechanistically, RNF186 acts as an E3 ubiquitin ligase that targets cytoplasmic HMGB1 for lysine 48 (K48)- and K63-linked ubiquitination, leading to its subsequent proteasomal degradation. Importantly, the translocation of HMGB1 from the nucleus to the cytoplasm is responsible for inducing lipophagy in NAFLD samples. Knockdown of HMGB1 significantly reduces the activation of lipophagy and mediates the decrease in lipid accumulation caused by RNF186 depletion in hepatocytes. Furthermore, we find that maintaining the nuclear HMGB1 level and inhibiting its nuclear-cytoplasmic shuttling are critical for the proper function of RNF186 in NAFLD. Additionally, the expression of RNF186 and HMGB1 in human NAFLD samples, along with factors related to lipophagy, suggest that RNF186 may play a similar role in the pathogenesis of human fatty liver. RNF186 deficiency accelerates hepatic lipophagy in NAFLD through the inhibition of ubiquitination and degradation of cytoplasmic HMGB1. Consequently, targeting the RNF186-HMGB1 axis may offer a promising strategy for the prevention and treatment of NAFLD. [Display omitted] • RNF186 regulates hepatic lipophagy in NAFLD. • RNF186 targets cytoplasmic HMGB1 for lysine 48 (K48)- and K63-linked ubiquitination and subsequent proteasomal degradation. • The nucleocytoplasmic translocation of HMGB1 is required for RNF186 function in NAFLD. [ABSTRACT FROM AUTHOR]

Abstract Background Steroidogenesis is an indispensable process that is indirectly associated with spermatogenesis in the Leydig cell (LC) to utilize the lipid droplets (LDs) that are critical to maintaining normal testosterone synthesis. The regulation of LD mobilization, known as lipophagy, in the LC is still largely unknown. Method In the present study, the LC of the Chinese soft-shelled turtle was investigated to identify the steroidogenic activity and lipophagy during the annual reproductive cycle by light microscopy, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM). Results The LC showed a dynamic steroidogenic function with strong activity of 3β-HSD, vimentin and tubular ER during hibernation by IHC and TEM. The tubulo-vesicular ER had a weak immunopositive reaction for 3β-HSD in the LC during reproductive phase, suggesting persistent steroidogenic activity. ORO staining and TEM demonstrated that a larger number of LDs had accumulated in the LC during hibernation than in the reproductive phase. These LDs existed in close association with mitochondria and lysosomes by being dynamically surrounded by intermediate filaments to facilitate LD utilization. Lysosomes were found directly attached to large LDs, forming an autophagic tube and engulfing LDs, suggesting that micro-lipophagy occurs during hibernation. Furthermore, the IHC of ATG7 (Autophagy Related Gene 7) and the IF of the LC3 (Microtubule-associated protein light chain 3), p62 (Sequestosome-1 (SQSTM1) and LAMP1(Lysosomal-associated membrane protein 1) results demonstrated strong expression, and further confirmation by TEM showed the existence of an autophagosome and an autolysosome and their fusion during the hibernation season. Conclusion In conclusion, the present study provides clear evidence of LD consumption in the LC by lipophagy, lysosome and mitochondria during the hibernation period, which is a key aspect of steroidogenesis in the Chinese soft-shelled turtle.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer’s disease. The principal pathological feature of PD is the progressive loss of dopaminergic neurons in the ventral midbrain. This pathology involves several cellular alterations: oxidative stress, mitochondrial dysfunction, loss of proteostasis, and autophagy impairment. Moreover, in recent years, lipid metabolism alterations have become relevant in PD pathogeny. The modification of lipid metabolism has become a possible way to treat the disease. Because of this, we analyzed the effect and possible mechanism of action of linoleic acid (LA) on an SH-SY5Y PD cell line model and a PD mouse model, both induced by 6-hydroxydopamine (6-OHDA) treatment. The results show that LA acts as a potent neuroprotective and anti-inflammatory agent in these PD models. We also observed that LA stimulates the biogenesis of lipid droplets and improves the autophagy/lipophagy flux, which resulted in an antioxidant effect in the in vitro PD model. In summary, we confirmed the neuroprotective effect of LA in vitro and in vivo against PD. We also obtained some clues about the novel neuroprotective mechanism of LA against PD through the regulation of lipid droplet dynamics.

(1) Background: The glucocorticoid receptor (GR) plays a key role in lipid metabolism, but investigations of GR activation as a potential therapeutic approach have been hampered by a lack of selective agonists. Ginsenoside compound K (CK) is natural small molecule with a steroid-like structure that offers a variety of therapeutic benefits. Our study validates CK as a novel GR agonist for the treatment of obesity. (2) Methods: By using pulldown and RNA interference, we determined that CK binds to GR. The anti-obesity potential effects of CK were investigated in obese mice, including through whole-body energy homeostasis, glucose and insulin tolerance, and biochemical and proteomic analysis. Using chromatin immunoprecipitation, we identified GR binding sites upstream of lipase ATGL. (3) Results: We demonstrated that CK reduced the weight and blood lipids of mice more significantly than the drug Orlistat. Proteomics data showed that CK up-regulated autophagy regulatory proteins, enhanced fatty acid oxidation proteins, and decreased fatty acid synthesis proteins. CK induced lipophagy with the initial formation of the phagophore via AMPK/ULK1 activation. However, a blockade of autophagy did not disturb the increase in CK on lipase expression, suggesting that autophagy and lipase are independent pathways in the function of CK. The pulldown and siRNA experiments showed that GR is the critical target. After binding to GR, CK not only activated lipophagy, but also promoted the binding of GR to the ATGL promoter. (4) Conclusions: Our findings indicate that CK is a natural food candidate for reducing fat content and weight.

Emerging evidence indicates that unexpected lipid droplet (LD) deposition and peroxidation can accelerate organelle stress and plays a crucial role in the pathogenesis of neurodegenerative diseases (NDDs). In our previous study, we confirmed that kaempferol (Ka), a natural flavonoid small molecule, exhibited neuroprotective effects on mice with LPS-induced Parkinson's disease (PD). In addition, previous studies have shown that autophagy plays an important role in the regulation of cellular LD deposition. In the current study, we showed that Ka protected against TH+ neuronal loss and behavioral deficits in MPTP/p-induced PD mice, accompanied by reduced lipid oxidative stress in the substantia nigra pars compacta (SNpc). In cultured neuronal cells, Ka exhibited a relatively safe concentration range and significantly suppressed LD accumulation and cellular apoptosis induced by MPP+. Further study indicated that the protective effect of Ka was dependent on autophagy, specifically lipophagy. Critically, Ka promoted autophagy to mediate LD degradation in lysosomes, which then alleviated lipid deposition and peroxidation and the resulting mitochondrial damage, consequently reducing neuronal death. Furthermore, AAV-shAtg5-mediated Atg5 knockdown abolished the neuroprotective effects of Ka against lipid oxidation in PD mice. This work demonstrates that Ka prevents dopaminergic neuronal degeneration in PD via the inhibition of lipid peroxidation-mediated mitochondrial damage by promoting lipophagy and provides a potential novel therapeutic strategy for PD and related NDDs.

Diseases caused by Flaviviridae have a wide global and economic impact due to high morbidity and mortality. Flaviviridae infection usually leads to severe, acute or chronic diseases, such as liver injury and liver cancer resulting from hepatitis C virus (HCV) infection, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) caused by dengue virus (DENV). Given the highly complex pathogenesis of Flaviviridae infections, they are still not fully understood at present. Accumulating evidence suggests that host autophagy is disrupted to regulate the life cycle of Flaviviridae. Organelle-specific autophagy is able to selectively target different organelles for quality control, which is essential for regulating cellular homeostasis. As an important sub process of autophagy, lipophagy regulates lipid metabolism by targeting lipid droplets (LDs) and is also closely related to the infection of a variety of pathogenic microorganisms. In this review, we briefly understand the LDs interaction relationship with Flaviviridae infection, outline the molecular events of how lipophagy occurs and the related research progress on the regulatory mechanisms of lipophagy in Flaviviridae infection. Exploring the crosstalk between viral infection and lipophagy induced molecular events may provide new avenues for antiviral therapy.

Diseases caused by Flaviviridae have a wide global and economic impact due to high morbidity and mortality. Flaviviridae infection usually leads to severe, acute or chronic diseases, such as liver injury and liver cancer resulting from hepatitis C virus (HCV) infection, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) caused by dengue virus (DENV). Given the highly complex pathogenesis of Flaviviridae infections, they are still not fully understood at present. Accumulating evidence suggests that host autophagy is disrupted to regulate the life cycle of Flaviviridae. Organelle-specific autophagy is able to selectively target different organelles for quality control, which is essential for regulating cellular homeostasis. As an important sub process of autophagy, lipophagy regulates lipid metabolism by targeting lipid droplets (LDs) and is also closely related to the infection of a variety of pathogenic microorganisms. In this review, we briefly understand the LDs interaction relationship with Flaviviridae infection, outline the molecular events of how lipophagy occurs and the related research progress on the regulatory mechanisms of lipophagy in Flaviviridae infection. Exploring the crosstalk between viral infection and lipophagy induced molecular events may provide new avenues for antiviral therapy. [ABSTRACT FROM AUTHOR]

Protein accumulation is the hallmark of various neuronal, muscular, and other human disorders. It is also often seen in the liver as a major protein‐secretory organ. For example, aggregation of mutated alpha1‐antitrypsin (AAT), referred to as PiZ, is a characteristic feature of AAT deficiency, whereas retention of hepatitis B surface protein (HBs) is found in chronic hepatitis B (CHB) infection. We investigated the interaction of both proteotoxic stresses in humans and mice. Animals overexpressing both PiZ and HBs (HBs‐PiZ mice) had greater liver injury, steatosis, and fibrosis. Later they exhibited higher hepatocellular carcinoma load and a more aggressive tumor subtype. Although PiZ and HBs displayed differing solubility properties and distinct distribution patterns, HBs‐PiZ animals manifested retention of AAT/HBs in the degradatory pathway and a marked accumulation of the autophagy adaptor p62. Isolation of p62‐containing particles revealed retained HBs/AAT and the lipophagy adapter perilipin‐2. p62 build‐up led to activation of the p62–Nrf2 axis and emergence of reactive oxygen species. Our results demonstrate that the simultaneous presence of two prevalent proteotoxic stresses promotes the development of liver injury due to protein retention and activation of the p62–Nrf2 axis. In humans, the PiZ variant was over‐represented in CHB patients with advanced liver fibrosis (unadjusted odds ratio = 9.92 [1.15–85.39]). Current siRNA approaches targeting HBs/AAT should be considered for these individuals. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

The autophagic degradation of lipid droplets (LDs), termed lipophagy, is a major mechanism that contributes to lipid turnover in numerous cell types. While numerous factors, including nutrient deprivation or overexpression of PNPLA2/ATGL (patatin-like phospholipase domain containing 2) drive lipophagy, the trafficking of fatty acids (FAs) produced from this pathway is largely unknown. Herein, we show that PNPLA2 and nutrient deprivation promoted the extracellular efflux of FAs. Inhibition of autophagy or lysosomal lipid degradation attenuated FA efflux highlighting a critical role for lipophagy in this process. Rather than direct transport of FAs across the lysosomal membrane, lipophagy-derived FA efflux requires lysosomal fusion to the plasma membrane. The lysosomal Ca2+ channel protein MCOLN1/TRPML1 (mucolipin 1) regulates lysosomal-plasma membrane fusion and its overexpression increased, while inhibition blocked FA efflux. In addition, inhibition of autophagy/lipophagy or MCOLN1, or sequestration of extracellular FAs with BSA attenuated the oxidation and re-esterification of lipophagy-derived FAs. Overall, these studies show that the well-established pathway of lysosomal fusion to the plasma membrane is the primary route for the disposal of FAs derived from lipophagy. Moreover, the efflux of FAs and their reuptake or subsequent extracellular trafficking to adjacent cells may play an important role in cell-to-cell lipid exchange and signaling. Abbreviations: ACTB: beta actin; ADRA1A: adrenergic receptor alpha, 1a; ALB: albumin; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; BECN1: beclin 1; BHBA: beta-hydroxybutyrate; BSA: bovine serum albumin; CDH1: e-cadherin; CQ: chloroquine; CTSB: cathepsin B; DGAT: diacylglycerol O-acyltransferase; FA: fatty acid; HFD: high-fat diet; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LIPA/LAL: lysosomal acid lipase A; LLME: Leu-Leu methyl ester hydrobromide; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryo fibroblast; PBS: phosphate-buffered saline; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA2/ATGL patatin-like phospholipase domain containing 2; RUBCN (rubicon autophagy regulator); SM: sphingomyelin; TAG: triacylglycerol; TMEM192: transmembrane protein 192; VLDL: very low density lipoprotein. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) or metabolic (dysfunction) associated liver disease (MAFLD), is, with a global prevalence of 25%, the most common liver disorder worldwide. NAFLD comprises a spectrum of liver disorders ranging from simple steatosis to steatohepatitis, fibrosis, cirrhosis and eventually end-stage liver disease. The cause of NAFLD is multifactorial with genetic susceptibility and an unhealthy lifestyle playing a crucial role in its development. Disrupted hepatic lipid homeostasis resulting in hepatic triglyceride accumulation is an hallmark of NAFLD. This disruption is commonly described based on four pathways concerning 1) increased fatty acid influx, 2) increased de novo lipogenesis, 3) reduced triglyceride secretion, and 4) reduced fatty acid oxidation. More recently, lipophagy has also emerged as pathway affecting NAFLD development and progression. Lipophagy is a form of autophagy (i.e. controlled autolysosomal degradation and recycling of cellular components), that controls the breakdown of lipid droplets in the liver. Here we address the role of hepatic lipid homeostasis in NAFLD and specifically review the current literature on lipophagy, describing its underlying mechanism, its role in pathophysiology and its potential as a therapeutic target.

Atherosclerosis, predominantly characterized by the disturbance of lipid homeostasis, has become the main causation of various cardiovascular diseases. Therefore, there is an urgent requirement to explore efficacious targets that act as lipid modulators for atherosclerosis. Transcription factor EB (TFEB), whose activity depends on post-translational modifications, such as phosphorylation, acetylation, SUMOylation, ubiquitination, etc., is significant for normal cell physiology. Recently, increasing evidence implicates a role of TFEB in lipid homeostasis, via its functionality of promoting lipid degradation and efflux through mediating lipophagy, lipolysis, and lipid metabolism-related genes. Furthermore, a regulatory effect on lipid transporters and lipid mediators by TFEB is emerging. Notably, TFEB makes a possible therapeutic target of atherosclerosis by regulating lipid metabolism. This review recapitulates the update and current advances on TFEB mediating lipid metabolism to focus on two intracellular activities: a) how cells perceive external stimuli and initiate transcription programs to modulate TFEB function, and b) how TFEB restores lipid homeostasis in the atherosclerotic process. In-depth research is warranted to develop potent agents against TFEB to alleviate or reverse the progression of atherosclerosis.

Atherosclerosis, predominantly characterized by the disturbance of lipid homeostasis, has become the main causation of various cardiovascular diseases. Therefore, there is an urgent requirement to explore efficacious targets that act as lipid modulators for atherosclerosis. Transcription factor EB (TFEB), whose activity depends on post-translational modifications, such as phosphorylation, acetylation, SUMOylation, ubiquitination, etc., is significant for normal cell physiology. Recently, increasing evidence implicates a role of TFEB in lipid homeostasis, via its functionality of promoting lipid degradation and efflux through mediating lipophagy, lipolysis, and lipid metabolism-related genes. Furthermore, a regulatory effect on lipid transporters and lipid mediators by TFEB is emerging. Notably, TFEB makes a possible therapeutic target of atherosclerosis by regulating lipid metabolism. This review recapitulates the update and current advances on TFEB mediating lipid metabolism to focus on two intracellular activities: a) how cells perceive external stimuli and initiate transcription programs to modulate TFEB function, and b) how TFEB restores lipid homeostasis in the atherosclerotic process. In-depth research is warranted to develop potent agents against TFEB to alleviate or reverse the progression of atherosclerosis. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) or metabolic (dysfunction) associated liver disease (MAFLD), is, with a global prevalence of 25%, the most common liver disorder worldwide. NAFLD comprises a spectrum of liver disorders ranging from simple steatosis to steatohepatitis, fibrosis, cirrhosis and eventually end-stage liver disease. The cause of NAFLD is multifactorial with genetic susceptibility and an unhealthy lifestyle playing a crucial role in its development. Disrupted hepatic lipid homeostasis resulting in hepatic triglyceride accumulation is an hallmark of NAFLD. This disruption is commonly described based on four pathways concerning 1) increased fatty acid influx, 2) increased de novo lipogenesis, 3) reduced triglyceride secretion, and 4) reduced fatty acid oxidation. More recently, lipophagy has also emerged as pathway affecting NAFLD development and progression. Lipophagy is a form of autophagy (i.e. controlled autolysosomal degradation and recycling of cellular components), that controls the breakdown of lipid droplets in the liver. Here we address the role of hepatic lipid homeostasis in NAFLD and specifically review the current literature on lipophagy, describing its underlying mechanism, its role in pathophysiology and its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis. [ABSTRACT FROM AUTHOR]

Lipid droplet (LD) degradation provides metabolic energy and important building blocks for various cellular processes. The two major LD degradation pathways include autophagy (lipophagy), which involves delivery of LDs to autolysosomes, and lipolysis, which is mediated by lipases. While abnormalities in LD degradation are associated with various pathological disorders, our understanding of lipophagy is still rudimentary. In this study, we describe the development of a lipophilic dye containing two fluorophores, one of which is pH-sensitive and the other pH-stable. We further demonstrate that this "Lipo-Fluddy" can be used to visualize and quantify lipophagy in living cells, in an easily applicable and protein label-free approach. After estimating the ability of compound candidates to penetrate LDs, we synthesized several BODIPY and (pH-switchable) rhodol dyes, whose fluorescence properties (incl. their photophysical compatibility) were analyzed. Of three Lipo-Fluddy dyes synthesized, one exhibited the desired properties and allowed observation of lipophagy by fluorescence microscopy. Also, this dye proved to be non-toxic and suitable for the examination of various cell lines. Moreover, a method was developed to quantify the lipophagy process using flow cytometry, which could be applied in the future in the identification of lipophagy-related genes or in the screening of potential drugs against lipophagy-related diseases., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

One means by which cells reutilize neutral lipids stored in lipid droplets is to degrade them by autophagy. This process involves spartin, mutations of which cause the rare inherited disorder Troyer syndrome (or spastic paraplegia-20, SPG20). A recently published paper from the team led by Karin Reinsich (Yale) suggests that the molecular function of spartin and its unique highly conserved "senescence" domain is as a lipid transfer protein. Spartin binds to and transfers all lipid species found in lipid droplets, from phospholipids to triglycerides and sterol esters. This lipid transfer activity correlates with spartin's ability to sustain lipid droplet turnover. The senescence domain poses an intriguing question around the wide range of its cargoes, but intriguingly it has yet to yield up its secrets because attempts at crystallization failed and AlphaFold's prediction is unconvincing., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)