Increased fatty acid synthesis and disturbed lipid metabolism in Neuro2a cells after repeated cocaine exposure: A preliminary study.

Chronic use of cocaine prompts neurodegeneration and neuroinflammation. Lipids play pivotal roles in neuronal function and pathology. Although evidence correlates cocaine use with the alteration of lipid metabolism in blood and brain, the precise mechanism remains to be elucidated. In this study, we explore the effect of cocaine on neuronal fatty acid profiles in vitro. Neuro2a cells following seven days of repeated exposure to cocaine (0, 600, 800, 1000 µM) showed apoptosis-irrelevant cell death, dysregulated autophagy, activation of atypical endoplasmic reticulum stress response, increased saturated and unsaturated fatty acid synthesis, and disrupted lipid metabolism. These preliminary findings indicated the association between lipid metabolism and cocaine-induced neurotoxicity, which should be beneficial for understanding the neurotoxicity of cocaine.

Alcohol and toxicological factors influencing fatal falls from height in the Greater Tokyo Area: a retrospective study.

Falls from height pose a significant public health concern in urban regions, including the highly urbanized Greater Tokyo Area. The Japanese population is characterized by high rates of suicide and psychoactive drug usage, underscoring the importance of investigating these attributes in falls from height. This study aimed to retrospectively analyze the alcohol and toxicological aspects influencing falls from height in the Greater Tokyo Area between 2014 and 2022 and compare the findings with existing reports on other populations. In total, 75 cases of falls from height and 159 cases of natural deaths were included. Consistent with previous findings, Fisher's exact test revealed a predominance of males (66.67%, 50/75) and young adults (57.33%, 43/75) in falls from height. Multivariate logistic regression analysis identified antidepressant usage as the most significant risk factor within the target population, while younger individuals under alcohol influence constituted another high-risk group. Notably, contradictory to other populations, female individuals involved in fatal falls in the Greater Tokyo Area exhibited a higher frequency of alcohol consumption than males (48.00%, 12/25 vs. 26.00%, 13/50), and most of them were associated with suicide (83.33%, 10/12). These findings elucidate the population characteristics that pose a high risk for fatal falls from height in Japan and can serve as a reference for other Asian populations residing in similar megacities.

Ectopic myogenesis and fibrosis accompany adipogenesis during thymic involution induced by repeated cocaine administration.

We have shown previously that daily cocaine administration for 7-14 days in rats resulted in the acceleration of thymic involution, which is, alike to age-related thymic involution, accompanied by ectopic adipogenesis. Here we show that the thymic involution caused by repeated cocaine administration is accompanied by not only adipogenesis but also ectopic myogenesis and fibrosis. In accordance with fibrosis, we observed an increase of N-cadherin, a marker of mesenchymal cells, as well as a decrease of E-cadherin, an epithelial cell marker, in the thymus in response to cocaine administration, suggesting the occurrence of epithelial-to-mesenchymal transition (EMT). Levels of fibronectin was also increased in the thymus of cocaine group compared to control group. In addition, increases in the levels of the transcription factors for myogenic differentiation, myogenin, MYF5, and MYF6, were observed in the thymus administered cocaine for 14 days. These results indicate that the thymic involution induced by cocaine administration involves not only adipogenesis and fibrosis but also ectopic myogenesis, which is scarcely observed and rather pathological process during thymic involution.

Excessive N-acetylcysteine exaggerates glutathione redox homeostasis and apoptosis during acetaminophen exposure in Huh-7 human hepatoma cells.

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway.

Induction of filamin-C and its involvement in the regulation of cellular senescence and apoptosis in Huh-7 hepatoma cells during arsenic trioxide exposure.

Arsenic trioxide (ATO) is one of the most toxic inorganic arsenic compounds. In this study, we examined the effects of long-term (7 days) exposure to low dose (5 µM) ATO on a human hepatocellular carcinoma cell line, Huh-7. Along with apoptosis accompanied by secondary necrosis though GSDME cleavage, we observed enlarged and flattened cells adhering to the culture dish and surviving even after exposure to ATO. An increase in cyclin-dependent kinase inhibitor p21 levels as well as positive staining for senescence-associated ß-galactosidase activity were observed in ATO-treated cells, indicating cellular senescence. Screening for both ATO-inducible proteins by MALDI-TOF-MS analysis and ATO-inducible genes by DNA microarray analysis showed a marked increase in filamin-C (FLNC), an actin cross-linking protein. Interestingly, the increase in FLNC was observed in both dead and surviving cells, suggesting that the upregulation of FLNC by ATO occurs in both apoptotic and senescent cells. Small interference RNA-mediated knock down of FLNC resulted in not only a reduction of senescence-associated enlarged morphology of the cells, but also an exacerbation of cell death. Taken together, these results suggest a regulatory role of FLNC in the execution of senescence as well as apoptosis during ATO exposure.

Cocaine induces vascular smooth muscle cells proliferation via DRP1-mediated mitochondrial fission and PI3K/HIF-1α signaling.

Long-term cocaine abuse is associated with cardiovascular and pulmonary vascular complications. The vascular toxicity of cocaine can lead to vascular remodeling characterized by excessive proliferation of vascular smooth muscle cells. Though hypoxia-inducible factor (HIF) signaling and mitochondrial fission have been suggested to play essential roles in the pathogenesis of hypoxia-induced vascular remodeling, pathogenetic mechanism for cocaine-related vascular remodeling remains to be elucidated. In this study, we explore the effect of cocaine on the proliferation of vascular smooth muscle cells by in vitro experiments. The findings indicated that the cocaine-induced vascular smooth muscle cell hyperproliferation is achieved by enhancing DRP1-mediated mitochondrial fission and activating PI3K/HIF-1α signaling. Current findings suggested that mitochondrial fission would play a pivotal role in cocaine-related vascular remodeling and would be helpful in understanding the vascular toxicity of cocaine.

Inverse regulation of GSDMD and GSDME gene expression during LPS-induced pyroptosis in RAW264.7 macrophage cells.

GSDMD and GSDME, members of the gasdermin protein family, are involved in the formation of plasma membrane channels contributing to cell rupture during a certain type of necrosis called pyroptosis. GSDMD is activated in response to immunological stimulation such as lipopolysaccharides (LPS) treatment while GSDME is mainly involved in drug-induced tumor cell death. Here we show that the expression of the GSDMD gene increases significantly during LPS-induced pyroptosis in RAW264.7 murine macrophage cells. In contrast, GSDME expression is decreased in the same cells. The increasing effect of LPS on GSDMD expression was observed only when the cells were cultured in high glucose (4.5 g/l) medium, suggesting that glucose availability is important for this effect. The effect of LPS on GSDMD expression is abolished by 2-deoxyglucose (2DG), confirming that glycolysis plays crucial roles in the increasing effect of LPS. Small interference RNA-mediated knock down of GSDMD or overexpression of GSDME causes LPS-induced pyroptosis to take place through GSDME rather than through GSDMD. Taken together, LPS regulates GSDMD and GSDME expression in opposite directions through, at least in part, its effect on glycolysis. This transcriptional regulation may contribute to the execution of pyroptosis in a GSDMD-dependent manner.

Role of Mitochondrial Dynamics in Cocaine's Neurotoxicity.

The dynamic balance of mitochondrial fission and fusion maintains mitochondrial homeostasis and optimal function. It is indispensable for cells such as neurons, which rely on the finely tuned mitochondria to carry out their normal physiological activities. The potent psychostimulant cocaine impairs mitochondria as one way it exerts its neurotoxicity, wherein the disturbances in mitochondrial dynamics have been suggested to play an essential role. In this review, we summarize the neurotoxicity of cocaine and the role of mitochondrial dynamics in cellular physiology. Subsequently, we introduce current findings that link disturbed neuronal mitochondrial dynamics with cocaine exposure. Finally, the possible role and potential therapeutic value of mitochondrial dynamics in cocaine neurotoxicity are discussed.

Contraction Band Necrosis with Dephosphorylated Connexin 43 in Rat Myocardium after Daily Cocaine Administration.

Contraction band necrosis (CBN) is a common abnormality found in the myocardium of cocaine abusers, but is rarely reported in experimental models of cocaine abuse. Connexin 43 (Cx43) is essential for cardiac intercellular communication and the propagation of CBN. Under stress or injury, cardiac Cx43 is dephosphorylated, which is related to cardiomyocyte dysfunction and pathogenesis, whereas adiponectin exerts beneficial effects in the myocardium. In this study, we explore the effects of cocaine on cardiac Cx43 in vivo. Rats were administered cocaine via the tail vein at 20 mg/kg/day for 14 days, and showed widespread CBN, microfocal myocarditis and myocardial fibrosis, corresponding to a dysfunction of cardiac mitochondria under increased oxidative stress. The increase in dephosphorylated cardiac Cx43 and its negative correlation with the myocardial distribution of CBN after cocaine administration were determined. In addition, apoptosis and necroptosis, as well as increased adiponectin levels, were observed in the myocardium after cocaine exposure. Accordingly, we found altered profiles of cardiac Cx43, CBN and its negative correlation with dephosphorylated cardiac Cx43, and the possible involvement of adiponectin in the myocardium after 14 days of cocaine administration. The latter might play a protective role in the cardiotoxicity of cocaine. The current findings would be beneficial for establishing novel therapeutic strategies in cocaine-induced cardiac consequences.

Extraglottic Airway Device Misplacement: A Novel Classification System and Findings in Postmortem Computed Tomography.

STUDY OBJECTIVE: Extraglottic airway devices are frequently used during cardiac arrest resuscitations and for failed intubation attempts. Recent literature suggests that many extraglottic airway devices are misplaced. The aim of this study is to create a classification system for extraglottic airway device misplacement and describe its frequency in a cohort of decedents who died with an extraglottic airway device in situ. METHODS: We assembled a cohort of all decedents who died with an extraglottic airway device in situ and underwent postmortem computed tomographic (CT) imaging at the state medical examiner's office during a 6-year period, using retrospective data. An expert panel developed a novel extraglottic airway device misplacement classification system. We then applied the schema in reviewing postmortem CT for extraglottic airway device position and potential complications. RESULTS: We identified 341 eligible decedents. The median age was 47.0 years (interquartile range 32 to 59 years). Out-of-hospital personnel placed extraglottic airway devices in 265 patients (77.7%) who subsequently died out of hospital; the remainder died inhospital. The classification system consisted of 6 components: depth, size, rotation, device kinking, mechanical blockage of ventilation opening, and injury. Under the system, extraglottic airway devices were found to be misplaced in 49 cases (14.4%), including 5 (1.5%) that resulted in severe injuries. CONCLUSION: We created a novel extraglottic airway device misplacement classification system. Misplacement occurred in greater than 14% of cases. Severe traumatic complications occurred rarely. Quality improvement activities should include review of extraglottic airway device placement when CT images are available and use the classification system to describe misplacements.

CT imaging of extraglottic airway device-pictorial review.

Compared to intubation with a cuffed endotracheal tube, extraglottic airway devices (EGDs), such as laryngeal mask airways, are considered less definitive ventilation conduit devices and are therefore often exchanged via endotracheal intubation (ETI) prior to obtaining CT images. With more widespread use and growing comfort among providers, reports have now described use of EGDs for up to 24 h including cases for which clinicians obtained CT scans with an EGD in situ. The term EGD encompasses a wide variety of devices with more complex structure and CT appearance compared to ETI. All EGDs are typically placed without direct visualization and require less training and time for insertion compared to ETI. While blind insertion generally results in functional positioning, numerous studies have reported misplacements of EGDs identified by CT in the emergency department or post-mortem. A CT-based classification system has recently been suggested to categorize these misplacements in six dimensions: depth, size, rotation, device kinking, mechanical blockage of the ventilation opening(s), and injury from EGD placement. Identifying the type of EGD and its correct placement is critically important both to provide prompt feedback to clinicians and prevent inappropriate medicolegal problems. In this review, we introduce the main types of EGDs, demonstrate their appearance on CT images, and describe examples of misplacements.

Increased circulating peroxiredoxin-4 in sepsis model rats involves secretion from hepatocytes and is mitigated by GYY4137.

Circulating peroxiredoxin-4 (Prx4) is suggested as a prognosis marker as well as a regulator of many diseases. We aimed to examine 1) whether Prx4 is secreted from the liver in an animal model of sepsis and 2) effects of GYY4137, a hydrogen sulfide donor molecule, on septic liver injury as well as the hepatic secretion of Prx4. Rats (Wistar, male, 6 weeks old) were administered lipopolysaccharide (LPS, 15 mg/kg body weight, i.p.) with or without pre-administration of GYY4137 (50 mg/kg body weight, i.p.) and sacrificed 24 h after LPS administration. Hematoxylin-eosin and Elastica Masson-Goldner stains were used to evaluate hepatic injuries. Cytokine expression levels were determined by qPCR, and the levels of Prx4 in the serum and liver were determined by immunoblotting. Hepatocytes were isolated from rat liver, and the levels of Prx4 in the medium as well as the cells were determined 24 h after the administrations of LPS (1 µg/ml), tumor necrosis factor-α (TNFα, 50 ng/ml), or interleukin-1ß (IL-1ß, 10 ng/ml), with or without GYY4137 (300 µM). Hepatic inflammation and damage in LPS-administered rats were suppressed by GYY4137. An increase in plasma Prx4 level caused by LPS was observed, but the increase was attenuated by pre-administration of GYY4137. Prx4 was secreted from isolated hepatocytes after stimulation with LPS, TNFα, or IL-1ß. GYY4137 attenuated the IL-1ß-induced Prx4 secretion from hepatocytes. Secretion from hepatocytes is likely involved in the increase in circulating Prx4 during sepsis. GYY4137 attenuates not only hepatic injury but also Prx4 secretion.

The down-regulation of cardiac contractile proteins underlies myocardial depression during sepsis and is mitigated by carbon monoxide.

The aim of this study is to investigate the mechanism underling cardiac dysfunction during sepsis, as well as the possible amelioration of this dysfunction by exogenous carbon monoxide (CO) administration. For this purpose, rats (six-week-old, male, Sprague-Dawley) were administered LPS (15 mg/kg body weight, i.p. 6 h) and/or CORM (30 mg/kg, i.p.). The decreased left ventricular ejection fraction (EF) observed in LPS group rats was recovered in the LSP + CORM group, confirming the protective role of CO against sepsis-induced myocardial depression. Proteomic as well as immunoblot analysis showed that the levels of myosin heavy and light chains (MHC and MLC) as well as α-cardiac actin (ACTC) were decreased in the LPS group, and these decreases were mitigated in the LSP + CORM group, suggesting that the amounts of major contractile proteins are decreased in depressed myocardium. Not only LPS-induced inflammatory cytokine (TNFα and IL-1ß) production but also the decrease in myofilament proteins was mitigated by CORM. These results confirm the protective action of exogenously administered CO against myocardial depression during sepsis, and reveal a novel mechanism underling cardiac dysfunction during sepsis.

Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation.

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells.

A CO-releasing molecule prevents annexin A2 down-regulation and associated disorders in LPS-administered rat lung.

To investigate septic lung injuries and the possible relief from injury by carbon monoxide (CO), rats were intraperitoneally (i.p.) administered water or the water-soluble CO-releasing molecule CORM (30 mg/kg body weight), followed by the successive administration of PBS or lipopolysaccharide (LPS, 15 mg/kg body weight, 6 h). The results in four experimental groups (control, LPS, LPS + CORM, CORM, n = 3 or 4 in each groups) were examined. Histological examination revealed the intravascular aggregation of erythrocytes in the lungs of the LPS group, and serological analysis showed a significant increase in D-dimer in the LPS group. Both the aggregation and D-dimer increase were ameliorated in the LPS + CORM group, suggesting that LPS-induced DIC in the lung is ameliorated by CORM. Proteomic as well as immunoblot analyses revealed that the levels of annexin A2 (AnxA2) were significantly decreased in the LPS group, but were at control levels in the LPS + CORM group. Concordant with the levels of AnxA2, the levels of both LC3 and collagen VI (COL VI) were decreased in the LPS group but not in the LPS + CORM group. Given the established roles of AnxA2 in fibrinolysis as well as intracellular vesicle trafficking, AnxA2 down-regulation should play an important role in the pathogenesis of septic lung injuries.

Ataxia telangiectasia and rad3 related (ATR)-promyelocytic leukemia protein (PML) pathway of the DNA damage response in the brain of rats administered arsenic trioxide.

To examine the in vivo responses of promyelocytic leukemia protein (PML) to arsenic, rats (male, 6 weeks old, Sprague Dawley) were administered a single intraperitoneal dose of 5 mg/kg arsenic trioxide (ATO). The protein was examined in the heart, lung, liver, and brain 6 and 48 hours after administration: a significant response of PML was observed in the brain. Oxidative DNA modification was also observed in the brain as revealed by increased immunoreactivity to anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody. In contrast, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain reactivity was only slightly increased, suggesting oxidative cellular stress without apoptotic cell death in the ATO-administered rat brain. Among the DNA damage response pathways, the ATR-Chk1 axis was activated, while the ATM-Chk2 axis was not, implying that the PML response is associated with activation of the ATR-Chk1 DNA repair pathway in the brain.

Increased MFG-E8 expression and its implications in the vascular pathophysiology of cocaine abuse.

The aim of this study was to examine the possible involvement of smooth muscle cell remodeling and the induction of MFG-E8 (milk fat globule protein epidermal growth factor-VIII) in vascular pathophysiology during cocaine administration in cultured cells and rats. Cocaine exerts bifurcate effects on vascular cells; it stimulates vasoconstriction through enhancement of catecholamine release at low doses, while it suppresses cardiovascular functions through inhibition of ion channels at high doses. Short-term exposure to a high concentration of cocaine (3 mM, 24 hr) resulted in cell death of A7r5 rat aorta-derived smooth muscle cells. On the other hand, long-term exposure of the same cells to a low concentration (0.3 mM, ~7 days) resulted in a transient increase in MFG-E8 expression followed by an increased tendency toward cyclin D1, PCNA (proliferating cell nuclear antigen), and CDK4 (cyclin-dependent protein kinase-4) expression. Interestingly, autophagy was not induced, but rather was impaired, in cocaine-treated cells. Increased expressions of MFG-E8, PCNA, and CDK4 were also observed in the aortic vascular cells of rats administered cocaine (50 mg/kg, 2 days, i.v.), confirming that cocaine induced MFG-E8 expression in vivo. Taken together, the results show that MFG-E8 is induced in vascular cells exposed to cocaine, and that this induction is likely to be involved in the vascular toxicity elicited by cocaine abuse.

Immobilization Stress With α2-Adrenergic Stimulation Induces Regional and Transient Reduction of Cardiac Contraction Through Gi Coupling in Rats.

Stress cardiomyopathy is characterized by transient apical hypokinesia related to catecholamine overflow. Recently, excessive epinephrine administration was shown to recapitulate stress cardiomyopathy through ß2-adrenoceptor (AR)-inhibitory G protein (Gi) coupling in rats. We aimed to study whether α2-AR and Gi affect cardiac contraction in rats in which emotional stress was evoked using immobilization (IMO). Echocardiography results showed that when male rats were exposed to IMO for 30 minutes and then injected with the α2-AR agonist xylazine (Xy), ejection fraction and the movement of the anterior wall (AW) were suppressed, maximally at 5 minutes post-injection, whereas posterior wall (PW) movement was preserved. At the same time points, the phosphorylation of Ser282 in myosin-binding protein-C (MyBP-C-Ser282) was higher in the PW than in the AW. Pretreatment with the Gi inhibitor pertussis toxin (PTX) reversed the low contractility and MyBP-C-Ser282 phosphorylation in the AW, but induced lethal heart failure in 3 out of 11 rats. Moreover, at 5 minutes after Xy injection following 30 minutes of IMO, serum epinephrine levels were increased. Thus, in rats exposed to psychological stress, α2-AR stimulation triggered transient hypo-contractility and MyBP-C-Ser282 hypo-phosphorylation in the AW, in association with an epinephrine surge. PTX treatment reversed the AW hypo-contractility and MyBP-C hypo-phosphorylation, but induced acute heart failure. These findings suggest α2AR/Gi-dependent signaling attenuates MyBP-C phosphorylation and contractility in the AW through an epinephrine surge in rats subjected to IMO and α2-AR stimulation. This model can recapitulate stress cardiomyopathy and thereby deepen our understanding of regional cardiac hypo-contractility and prosurvival mechanisms.

Lipopolysaccharide induces expression of collagen VI in the rat lung.

The involvement of the lung during the septic systemic inflammatory response elicited by administration of lipopolysaccharide (LPS) was investigated. Eight-week-old male Sprague-Dawley rats were injected i.p. with 15 mg/kg LPS. After 24 h, the lungs were excised to evaluate the cellular responses to LPS. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis revealed that type VI collagen (ColVI) was extremely upregulated during sepsis in the rat lung within the first 24 h of LPS administration. Upregulation of ColVI protein and its mRNA was demonstrated by Western blot analysis, real time PCR, and immunohistochemistry. To the best of our knowledge, this is the first report demonstrating the activation of ColVI in the rat lung at the early stage of systemic inflammation. Activation of ColVI might be involved in sepsis-mediated lung fibrosis at an early stage.

Immunohistochemical study of the autophagy marker microtubule-associated protein 1 light chain 3 in normal and steatotic human livers.

AIM: Autophagy has been implicated in lipid droplet (LD) turnover. Adipose differentiation-related protein (ADRP) and microtubule-associated protein 1 light chain 3 (LC3) monitor LD and autophagosomes, respectively. We examined whether immunohistochemical staining of ADRP and LC3 can monitor LD and autophagy, and if so, whether autophagy is related to LD turnover in post-mortem human livers. METHODS: We performed conventional immunohistochemistry of LC3 in paraffin-embedded human livers with different severities of steatosis, obtained at autopsy. Double immunofluorescence microscopy using anti-LC3 and anti-ADRP antibodies was performed to elucidate the relationship between autophagy and LD turnover. RESULTS: LC3 immunohistochemistry reproducibly delineated puncta in normal human livers, which were preferentially located around the central venal zone. The extent of LC3 immunostaining reduced with progressing steatosis. Double immunofluorescence for ADRP and LC3 demonstrated an inverse relationship between ADRP positive areas and LC3 positive areas, as well as the co-localization of ADRP and LC3 on a part of small LD but not large LD. CONCLUSION: These findings suggest that impaired autophagy promotes steatosis and that autophagy may be implicated in LD turnover.