Increased lipogenesis and lipidosis of gallbladder epithelium in dogs with gallbladder mucocele formation.

BACKGROUND: Gallbladder disease in people is frequently associated with disorders of lipid metabolism and metabolic syndrome. A recently emergent gallbladder disease of dogs, referred to as mucocele formation, is characterized by secretion of abnormal mucus by the gallbladder epithelium and is similarly associated with hyperlipidemia, endocrinopathy, and metabolic dysfunction. The cause of gallbladder mucocele formation in dogs is unknown. METHODS: A prospective case-controlled study was conducted to gain insight into disease pathogenesis by characterization of plasma lipid abnormalities in 18 dogs with gallbladder mucocele formation and 18 age and breed matched control dogs using direct infusion mass spectrometry for complex plasma lipid analysis. This analysis was complemented by histochemical and ultrastructural examination of gallbladder mucosa from dogs with gallbladder mucocele formation and control dogs for evidence of altered lipid homeostasis of the gallbladder epithelium. RESULTS: Gallbladder mucocele formation in dogs carried a unique lipidomic signature of increased lipogenesis impacting 50% of lipid classes, 36% of esterified fatty acid species, and 11% of complex lipid species. Broad enrichment of complex lipids with palmitoleic acid (16:1) and decreased abundance within complex lipids of presumptive omega-3 fatty acids eicosapentaenoic (20:5) and docosahexaenoic (22:6) was significant. Severe lipidosis of gallbladder epithelium pinpoints the gallbladder as involved causally or consequently in abnormal lipid metabolism. CONCLUSION: Our study supports a primary increase in lipogenesis in dogs with mucocele formation and abnormal gallbladder lipid metabolism in disease pathogenesis.

[The lipidosis of the liver of dairy cows: Part 1 - Role of insulin and the Growth Hormone-IGF-1 axis]. / Die Leberverfettung der Milchkuh: Teil 1.

Lipidosis of the liver of dairy cows is a metabolic disease known since many years and is caused by an uptake of nonesterified fatty acids (NEFA) into the liver cells, limited metabolism of NEFA (oxidation and production of ß-hydroxybutyrate), and resynthesis in relation to a low efflux as triglyceride (TG). The pathogenesis of lipidosis includes a) an augmented release of NEFA by mobilisation of adipose tissue, b) uptake of NEFA into the liver cells, c) metabolism of NEFA and d) re-synthesis of triglyceride and e) an efflux of TG as very low density lipoprotein (VLDL). The steps a-e are postpartum modified by hormones as an increase of growth hormone, a pronounced insulin resistance in combination with a decreased insulin and of IGF-1 concentrations. These hormonal changes are related to an uncoupling of the growth hormone-IGF-1-axis with enhanced lipolysis and consequences mentioned above. These alterations are associated with inflammation, oxidative and endoplasmatic stress. The metabolic and hormonal alterations are the result of the selection of dairy cows primarily for milk production without adequate food intake with the consequence of lipidosis, ketosis and further health risks (production diseases).

Hepatic Lipidosis in Ruminants.

Hepatic lipidosis (ie, fatty liver) occurs primarily during the first weeks of lactation in dairy cows because of excessive lipolysis overwhelming the concomitant capacity for beta-oxidation and hepatic export of triglycerides. Besides economic losses due to reduced lactational and reproductive performance, close associations with concomitantly occurring infectious and metabolic health disorders, in particular ketosis, exist. Hepatic lipidosis is not only a consequence from the postpartal negative energy balance but also acts as a disease component for further health disorders.

Associations between ultrasound measurements and hematochemical parameters for the assessment of liver metabolic status in Holstein-Friesian cows.

Metabolic disorders, including hepatic lipidosis and ketosis, severely affect animal health status and welfare with a large economic burden in dairy herds. The gold standard for diagnosing hepatic lipidosis is the liver biopsy, which is impractical and invasive for the screening at farm level. Ultrasound (US) imaging is a promising technique for identifying liver dysfunction, but standardized specifications in physiological conditions are needed. Herein, we described the features of four US measurements, namely the liver predicted triacylglycerol (pTAG) content, liver depth (LD), and portal vein area (PVA) and depth (PVD) and we investigated their associations with a set of hematochemical (HC) indicators in 342 clinically healthy Holstein Friesian dairy cows. Liver pTAG content was negatively associated with hematocrit and positively with globulin, whereas PVA was negatively associated with thiol group levels, and LD positively with ceruloplasmin. We found significant interactions between some HC parameters and parity: in particular, creatinine, thiol groups and globulin for PVA, and aspartate aminotransferase, paraoxonase and ceruloplasmin for PVD. This study offers new insights on variations in liver function occurring after calving and pave the way for the potential use of minimally invasive techniques for prompt detection of metabolic disorders in dairy herds.

Acarbose has sex-dependent and -independent effects on age-related physical function, cardiac health, and lipid biology.

With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat type 2 diabetes mellitus (T2DM), can extend mouse life span with greater effect in males than in females. Using a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological, and biochemical changes that occur during aging, and we determined which of the effects of age and drug were sex dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength test. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth-promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids - changes that have previously been associated with aging, cardiac dysfunction, and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging antiaging effects in multiple tissues, and it may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.

Consumption of a high energy density diet triggers microbiota dysbiosis, hepatic lipidosis, and microglia activation in the nucleus of the solitary tract in rats.

INTRODUCTION: Obesity is a multifactorial chronic inflammatory disease. Consumption of high energy density (HED) diets is associated with hyperphagia, increased body weight and body fat accumulation, and obesity. Our lab has previously shown that short-term (4 weeks) consumption of a HED diet triggers gut microbiota dysbiosis, gut inflammation, and reorganization of the gut-brain vagal communication. OBJETIVES: The aim of this study was to investigate the effect of long-term (6 months) consumption of HED diet on body composition, gut microbiome, hepatocellular lipidosis, microglia activation in the nucleus of the solitary tract, and systemic inflammation. METHODS: Male Sprague-Dawley rats were fed a low energy density (LED) diet for 2 weeks and then switched to a HED diet for 26 weeks. Twenty-four-hour food intake, body weight, and body composition were measured twice a week. Blood serum and fecal samples were collected at baseline, 1, 4, 8, and 26 weeks after introduction of the HED diet. Serum samples were used to measure insulin, leptin, and inflammatory cytokines using Enzyme-linked Immunosorbent Assay. Fecal samples were assessed for 16 S rRNA genome sequencing. RESULTS: HED diet induced microbiota dysbiosis within a week of introducing the diet. In addition, there was significant microglia activation in the intermediate NTS and marked hepatic lipidosis after 4 weeks of HED diet. We further observed changes in the serum cytokine profile after 26 weeks of HED feeding. CONCLUSIONS: These data suggest that microbiota dysbiosis is the first response of the organism to HED diets, followed by increased liver fat accumulation, microglia activation in the brain, and circulating levels of inflammatory markers. To our knowledge, this is the first study to present longitudinal and cross-sectional results on effect of long-term consumption of HED diets on all these parameters in a single cohort of animals.

Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis.

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

Gene expressions of de novo hepatic lipogenesis in feline hepatic lipidosis.

OBJECTIVES: The aim of this study was to evaluate if de novo hepatic lipid synthesis contributes to fatty acid overload in the liver of cats with feline hepatic lipidosis (FHL). METHODS: Lipogenic gene expression of peroxisome proliferator-activated receptor-alpha (PPAR-α), peroxisome proliferator-activated receptor-gamma (PPAR-γ), fatty acid synthase (FASN) and sterol regulatory element-binding factor (SREBF1) were evaluated using quantitative RT-PCR in liver tissue of six cats with FHL and compared with the liver tissue of eight healthy cats. RESULTS: In liver tissue, PPAR-α, PPAR-γ and FASN mRNA expression levels were not significantly different (P >0.12, P >0.89 and P >0.5, respectively) in the FHL group compared with the control group. SREBF1 gene expression was downregulated around 10-fold in the FHL group vs the control group (P = 0.039). CONCLUSIONS AND RELEVANCE: The downregulation of SREBF1 in the liver tissue of cats with FHL does not support the hypothesis that de novo lipogenesis in the liver is an important pathway of fatty acid accumulation in FHL.

Protective effects of sulforaphane on type 2 diabetes-induced cardiomyopathy via AMPK-mediated activation of lipid metabolic pathways and NRF2 function.

BACKGROUND: AMP-activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. It was reported that sulforaphane (SFN) prevented type 2 diabetes (T2D)-induced cardiomyopathy accompanied by the activation of AMPK; In this study, AMPK's pivotal role in SFN-mediated prevention against T2D-induced cardiomyopathy was tested using global deletion of AMPKα2 gene (AMPKα2-KO) mice. METHODS AND RESULTS: T2D was established by feeding 3-month high-fat diet (HFD) to induce insulin resistance, followed by an intraperitoneal injection of streptozotocin (STZ) to induce mild hyperglycemia in both AMPKα2-KO and wild-type (WT) mice. Then both T2D and control mice were subsequently treated with or without SFN for 3 months while continually feeding HFD or normal diet. Upon completion of the 3-month treatment, five mice from each group were sacrificed as a 3-month time-point (3 M). The rest continued normal diet or HFD until terminating study at the sixth month (6 M) of diabetes. Cardiac function was examined with echocardiography before sacrifice at both 3 M and 6 M. SFN prevented T2D-induced progression of cardiac dysfunction, remodeling (hypertrophy and fibrosis), inflammation, and oxidative damage in wild-type diabetic mice, but not in AMPKα2-KO mice. Mechanistically, SFN prevented T2D-induced cardiomyopathy not only by improving AMPK-mediated lipid metabolic pathways, but also enhancing NRF2 activation via AMPK/AKT/GSK3ß pathway. However, these improving effects of SFN were abolished in AMPKα2-KO diabetic mice. CONCLUSIONS: AMPK is indispensable for the SFN-induced prevention of cardiomyopathy in T2D, and the activation of NRF2 by SFN is mediated by AMPK/AKT/GSK3ß signaling pathways.

Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

BACKGROUND: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. OBJECTIVE: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. ANIMALS: Liver organoids obtained from 6 cats. METHODS: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. RESULTS: Both T863 (a diacylglycerol O-acyltransferase 1 [DGAT1] inhibitor) and 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P < .0001) and 46% (P = .0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. CONCLUSIONS AND CLINICAL IMPORTANCE: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid-lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species-specific system and provide the basis for further clinical testing of drugs to treat steatosis.

PPAR δ inhibition protects against palmitic acid-LPS induced lipidosis and injury in cultured hepatocyte L02 cell.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and its pathogenesis and mechanism are intricate. In the present study, we aimed to evaluate the role of PPAR δ in LPS associated NAFLD and to investigate the signal transduction pathways underlying PPAR δ treatment in vitro. Material and Methods: L02 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of PPAR δ inhibition and/or activation. Results: LPS treatment markedly increased lipid deposition, FFA contents, IL-6 and TNF-α levels, and cell apoptosis in PA treatment (NAFLD model). PPAR δ inhibition protects L02 cells against LPS-induced lipidosis and injury. Conversely, the result of PPAR δ activation showed the reverse trend. LPS+PA treatment group significantly decreases the relative expression level of IRS-1, PI3K, AKT, phosphorylation of AKT, TLR-4, MyD88, phosphorylation of IKKα, NF-κB, Bcl-2 and increases the relative expression level of Bax, cleaved caspase 3 and cleaved caspase 8, compared with the cells treated with NAFLD model. PPAR δ inhibition upregulated the related proteins' expression level in insulin resistance and inflammation pathway and downregulated apoptotic relevant proteins. Instead, PPAR δ agonist showed the reverse trend. Conclusion: Our data show that PPAR δ inhibition reduces steatosis, inflammation and apoptosis in LPS-related NAFLD damage, in vitro. PPAR δ may be a potential therapeutic implication for NAFLD.

Amino acid pattern in the liver and blood of fattening turkeys suffering from hepatic lipidosis.

Hepatic lipidosis (HL) is a well-known disease in fattening and in parent turkey flocks. Among others, dietary effects like (a lack of) essential amino acids (AA) as lipotrophic factors (e.g., methionine) have been considered as potentially predispositing for HL. Several studies have reported abnormal AA profiles in hepatic diseases of humans and other livestock. The ratio of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) in plasma is used to predict hepatic cirrhosis. In this study, the state of supply of AA was investigated by comparing non-affected (NA) animals and those affected by HL. The AA pattern in the liver and blood can provide potential indications of pathogenesis of HL. In cooperation with German poultry veterinarians, 3 cases of HL on 3 different fattening turkey farms were visited (13/14 wk old, "B.U.T. Big 6" and "TP7"). Overall, 73 birds were examined, of which 42 birds suffered from HL and 31 were not affected. Feeding samples of the respective actual feed were taken and analyzed. The selection of animals was carried out (NA randomly) by clinical signs such as apathy and dyspnea and the diagnosis was made at necropsy, which could be confirmed by crude fat content in liver tissue (HL: 309, NA: 155). In liver tissue, the CP and AA contents were lower among animals with HL than among NA (P < 0.05). In blood samples, the sum of AA, ammonia, and urea was more than 3 times higher among animals with HL (431 mg/dL serum) than among NA (114 mg/dL serum; P < 0.01). The ratio of BCAA to AAA was also significantly different between the groups (HL: 0.85, NA: 1.42; P < 0.05). In the case of HL, entire herds were not affected and the "non-affected" ones were comparable with healthy slaughtered animals. There seems to be a clear change in protein and AA metabolism of HL animals, which could lead to an optimization in feeding practice in repeated cases of HL.

Optic Nerve Lipidomics Reveal Impaired Glucosylsphingosine Lipids Pathway in Glaucoma.

Purpose: To determine major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Methods: Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extracted using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), respectively. Lipids were extracted after weighing the tissue; the protein amounts in the corresponding aqueous phase of organic solvent extraction were recorded. High-resolution mass spectrometry was performed using a Q-exactive mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph instrument. Bioinformatics and statistical analysis were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amounts were determined using Bradford's method. Western blot, ELISA, and immunohistochemistry utilized established protocols and were performed for protein quantification and localization, respectively. Additional donor tissues were utilized for Western blot, ELISA, and immunohistochemistry. Results: Principal component analysis (PCA) placed control and glaucomatous ONs in two distinct groups based on analysis of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. Conclusions: We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymatic pathways in lysosomal and nonlysosomal cellular compartments.

Altered lipoprotein profiles in cats with hepatic lipidosis.

OBJECTIVES: The aim of this study was to assess serum lipoprotein profiles using rapid single-spin continuous lipoprotein density profiling (CLPDP) in healthy control cats and cats with hepatic lipidosis (HL). METHODS: Analysis of serum lipoprotein profiles using the CLPDP was performed in 23 cats with HL and 20 healthy control cats. The area under the curve for each lipoprotein fraction, triglyceride (TG)-rich lipoproteins (TRLs), low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs), was calculated. Serum cholesterol and TG concentrations were measured using a clinical chemistry analyzer. RESULTS: Serum cholesterol and TG concentrations were not significantly different between healthy control cats and cats with HL ( P = 0.5075 and P = 0.2541, respectively). LDL content was significantly higher in cats with HL than in healthy control cats ( P = 0.0001), while HDL content was significantly lower in cats with HL than in healthy control cats ( P = 0.0032). TRL content was not significantly different between the two groups ( P = 0.0699). The specific fraction (1.037-1.043 g/ml) within nominal LDL in serum distinguished healthy control cats from cats with HL with a sensitivity of 87% and a specificity of 90%. CONCLUSIONS AND RELEVANCE: Serum lipoprotein profiles were altered in cats with HL, even though serum cholesterol and TG concentrations were not significantly different compared with healthy control cats. The CLPDP might be a useful tool for assessing lipid metabolism in cats with HL.

Topiramate protects apoE-deficient mice from kidney damage without affecting plasma lipids.

Topiramate is an anticonvulsant drug also prescribed for migraine prophylaxis that acts through several mechanisms of action. Several studies indicate that topiramate induces weight loss and a moderate reduction of plasma lipids and glucose. Based on these favourable metabolic effects, aim of this study was to evaluate if topiramate could modulate atherosclerosis development and protect target organs of dysmetabolic conditions. Thirty apoE-deficient mice were divided into three groups and fed for 12 weeks a high fat diet (Control) or the same diet containing topiramate at 0.125% and 0.250%. Body weight, water and food intake were monitored throughout the study. Plasma lipids and glucose levels were measured and a glucose tolerance test was performed. Atherosclerosis development was evaluated in the whole aorta and at the aortic sinus. Histological analysis of liver, kidney and adipose tissue was performed. Topiramate did not affect weight gain and food intake. Glucose tolerance and plasma lipids were not changed and, in turn, atherosclerosis development was not different among groups. Topiramate did not modify liver and adipose tissue histology. Conversely, in the kidneys, the treatment reduced the occurrence of glomerular lipidosis by decreasing foam cells accumulation and reducing the expression of inflammatory markers. Blood urea nitrogen levels were also reduced by treatment. Our results indicate that topiramate does not affect atherosclerosis development, but preserves kidney structure and function. The study suggests that topiramate could be investigated in drug repurposing studies for the treatment of glomerular lipidosis.

Pathological Features of Corneal Phospholipidosis in Juvenile White Rabbits Induced by Ocular Instillation of Chloroquine or Amiodarone.

Cationic amphiphilic drugs (CADs) can induce phospholipidosis (PLD) in organs/tissues. Several ophthalmic pharmaceuticals containing CADs are marketed and used in children. To investigate the effect of PLD on the developing cornea, chloroquine and amiodarone, which are representative CADs, were applied topically to the eyes of juvenile rabbits, and the effects in juvenile rabbits were compared with those in young adult rabbits. Diffuse corneal cloudiness was observed in chloroquine- and amiodarone-treated eyes. Histopathologically, vacuolation was observed in the corneal epithelium and keratocytes. On ultrastructural examination, these vacuoles contained multilamellar inclusion bodies, which are a characteristic of PLD. The size of the vacuoles in the corneal epithelium was reduced in juveniles compared with young adults. Cytoplasmic lamellar bodies and exocytosis in the corneal endothelium were observed in young adult rabbits but not in juvenile rabbits. This study revealed that topical application of chloroquine or amiodarone induces corneal PLD in juvenile and young adult rabbits. Corneal endothelial changes occurred only in young adult rabbits, but ophthalmological changes were similar between juveniles and young adults. The results of the study suggest that the effects of corneal PLD were similar among age groups based on risk assessment.

Imaging Mass Microscopy of Kidneys from Azithromycin-Treated Rats with Phospholipidosis.

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of tissue phospholipids. Although azithromycin can be used to induce phospholipidosis, no experimental studies evaluating the relationship between drug accumulation and phospholipid localization have been performed. In this study, azithromycin was orally administered to rats for 7 days, and the relationship between drug and phospholipid accumulation was performed using imaging mass microscopy. The administration of azithromycin induced tubular epithelial vacuolation in the inner stripe of the outer medulla of the kidney, consistent with the lamellar bodies that are typical manifestations of drug-induced phospholipidosis. Azithromycin and phospholipid tissue levels were extensively elevated in the kidneys of azithromycin-treated rats. Imaging mass microscopy revealed that both azithromycin and its metabolites were found in the kidneys of azithromycin-treated rats but not in control animals. The vacuolated areas of the kidneys were primarily found in the inner stripe of the outer medulla, consistent with the areas of high azithromycin concentration. Azithromycin was colocalized with several phospholipids-phosphatidylinositol (18:0/20:4), phosphatidylethanolamine (18:0/20:4 and 16:0/20:4), and possibly didocosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate, a putative biomarker of drug-induced phospholipidosis. In summary, we found correlations between regions of kidney damage and the accumulation of azithromycin, its metabolites, and phospholipids using imaging mass microscopy. Such analyses may help reveal the mechanism and identify putative biomarkers of drug-induced phospholipidosis.

Ectopic Lipid Deposition Is Associated With Insulin Resistance in Postmenopausal Women.

Context: Menopause is associated with an increased incidence of insulin resistance and diabetes. Objective: The aim of this study was to explore the lipid deposition in liver and skeletal muscle and investigate the association with insulin sensitivity in postmenopausal and premenopausal women. Design and Setting: Single-center cross-sectional study of 55 healthy women between 45 and 60 years of age. We measured lipid deposition in the liver with magnetic resonance spectroscopy, intramuscular and intra-abdominal lipid deposition with MRI, body composition with a dual-energy X-ray absorptiometry scan, and insulin sensitivity with the composite Matsuda Index. Outcome Measures: We studied the association between fat distribution, ectopic lipid deposition, and insulin sensitivity in pre- and postmenopausal women. Results: Postmenopausal women had an increased lipid deposition in the liver [0.68% (0.44 to 0.99) vs 0.49% (0.38 to 0.64), P = 0.01] and skeletal muscle [3% (2 to 4) vs 2% (1 to 3), P = 0.001] and had a 28% lower Matsuda insulin sensitivity index during an oral glucose tolerance test (6.31 ± 3.48 vs 8.78 ± 4.67, P = 0.05) compared with premenopausal women. Total fat mass and leg fat mass were stronger predictors of ectopic lipid deposition, and visceral fat mass was a stronger predictor of both ectopic lipid deposition and insulin resistance in postmenopausal women compared with premenopausal women. Conclusions: For a given subcutaneous and visceral fat depot size, postmenopausal women show increased ectopic lipid deposition and insulin resistance compared with premenopausal women. It is suggested that lipid deposition in liver and skeletal muscle may represent important mechanistic links between the changes in fat depots and the increased incidence of insulin resistance seen after menopause.

Assessment of amiodarone-induced phospholipidosis in chimeric mice with a humanized liver.

It is important to consider susceptibility to drug-induced toxicity between animals and humans. Chimeric mice with a humanized liver are expected to predict hepatotoxicity in humans. Drug-induced phospholipidosis (DIPL), in which phospholipids accumulate, is a known entity. In this study, we examined whether chimeric mice can reveal species differences in DIPL. Changes in various phosphatidylcholine (PhC) molecules were investigated in the liver of chimeric mice after administering amiodarone, which induces phospholipidosis. Liquid chromatography-tandem mass spectrometry revealed that levels of PhCs tended to increase in the liver after administration of amiodarone. The liver of chimeric mice consists of human hepatocytes and residual mouse hepatocytes. We used imaging mass spectrometry (IMS) to evaluate the increase of PhCs in human and mouse hepatocytes after administration of amiodarone. IMS visualizes localization of endogenous and exogenous molecules in tissues. The IMS analysis suggested that the localized levels of several PhCs tended to be higher in the human hepatocytes than those in mouse hepatocytes, and PhC levels changed in response to amiodarone. Chimeric mice with a humanized liver will be useful to evaluate species differences in DIPL between mice and humans.

A cell-based assay using HepaRG cells for predicting drug-induced phospholipidosis.

The utility of HepaRG cells as an in vitro cell-based assay system for predicting drug-induced phospholipidosis (PLD) was investigated. In experiment 1, 10 PLD-positive compounds and 11 PLD-negative compounds were selected. HepaRG cells were treated with each compound for 48 hr. In experiment 2, loratadine and desloratadine, a major metabolite of loratadine, were used to assess metabolic activation for PLD. HepaRG cells were treated with loratadine and desloratadine in the presence or absence of 500 µM 1-aminobenzotriazole (ABT), a broad CYP inhibitor, for 48 hr. After treatment with compounds in experiments 1 and 2, the relative fluorescence intensity (RFI) was measured using LYSO-ID Red dye to assess the PLD induction. In experiment 1, our cell-based assay system using HepaRG cells exhibited 100% sensitivity and 100% specificity for predicting drug-induced PLD. In experiment 2, loratadine increased the RFI in the PLD assay. However, the increase in the RFI was not observed in co-treatment with loratadine and ABT. In addition, desloratadine increased the RFI in the presence and absence of ABT. These results suggested that metabolic activation of loratadine may contribute to PLD in HepaRG cells. We newly demonstrated that HepaRG cells have a high ability for predicting drug-induced PLD. In addition, we newly showed that HepaRG cells may predict drug-induced PLD mediated by metabolic activation of loratadine. Thus, a cell-based assay system using HepaRG cells is a useful model for predicting drug-induced PLD.