Low Serum Lysosomal Acid Lipase Activity Correlates with Advanced Liver Disease

Fatty liver has become the most common liver disorder and is recognized as a major health burden in the Western world. The causes for disease progression are not fully elucidated but lysosomal impairment is suggested. Here we evaluate a possible role for lysosomal acid lipase (LAL) activity in liver disease. To study LAL levels in patients with microvesicular, idiopathic cirrhosis and nonalcoholic fatty liver disease (NAFLD). Medical records of patients with microvesicular steatosis, cryptogenic cirrhosis and NAFLD, diagnosed on the basis of liver biopsies, were included in the study. Measured serum LAL activity was correlated to clinical, laboratory, imaging and pathological data. No patient exhibited LAL activity compatible with genetic LAL deficiency. However, serum LAL activity inversely predicted liver disease severity. A LAL level of 0.5 was the most sensitive for detecting both histologic and noninvasive markers for disease severity, including lower white blood cell count and calcium, and elevated γ-glutamyltransferase, creatinine, glucose, glycated hemoglobin, uric acid and coagulation function. Serum LAL activity <0.5 indicates severe liver injury in patients with fatty liver and cirrhosis. Further studies should define the direct role of LAL in liver disease severity and consider the possibility of replacement therapy.     

Characterization of the adipose tissue atrophy induced by peroxisome proliferators in mice

In the present study, we characterized the effects of peroxisome proliferators (PP) on adipose tissue in mice. Treatment with potent PP, such as perfluorooctanoic acid (PFOA), 2-methyl-2-(p(1,2,3,4-tetrahydroxy-naphthyl)-phenoxy)propionic acid, (4-chloro-6-(2,3-xylidino)2-pyrimidinylthio) acetic acid, and di(2-ethylhexyl)phthalate, caused dramatic decreases in adipose tissue weight, whereas the moderately potent PP, acetylsalicylic acid, had a relatively weak effect. This decrease in weight reflects a loss of fat from adipocytes rather than a loss of cells, as demonstrated by constant DNA content. The dose-dependency and time-course experiments indicate that peroxisome proliferation occurs simultaneously with or prior to adipose tissue atrophy. Thus, hepatic peroxisome proliferation might result in the increased mobilization of lipids and lipid utilization in liver. The enhanced adipose tissue hormonesensitive lipase (HSL) activity and down-regulated lipoprotein lipase (LPL) activity observed upon PP treatment might, at least in part, explain the loss of fat via increase FA release from adipocytes and/or decreased FA uptake from the circulation, respectively. In addition, the possible involvement of the increased tumor necrosis factor α expression found upon PFOA treatment in reducing the insulin sensitivity of adipose tissue and thereby altering LPL and HSL activities is discussed.     

Does Lysosomial Acid Lipase Reduction Play a Role in Adult Non-Alcoholic Fatty Liver Disease?

Lysosomal Acid Lipase (LAL) is a key enzyme involved in lipid metabolism, responsible for hydrolysing the cholesteryl esters and triglycerides. Wolman Disease represents the early onset phenotype of LAL deficiency rapidly leading to death. Cholesterol Ester Storage Disease is a late onset phenotype that occurs with fatty liver, elevated aminotransferase levels, hepatomegaly and dyslipidaemia, the latter characterized by elevated LDL-C and low HDL-C. The natural history and the clinical manifestations of the LAL deficiency in adults are not well defined, and the diagnosis is often incidental. LAL deficiency has been suggested as an under-recognized cause of dyslipidaemia and fatty liver. Therefore, LAL activity may be reduced also in non-obese patients presenting non-alcoholic fatty liver disease (NAFLD), unexplained persistently elevated liver transaminases or with elevation in LDL cholesterol. In these patients, it could be indicated to test LAL activity. So far, very few studies have been performed to assess LAL activity in representative samples of normal subjects or patients with NAFLD. Moreover, no large study has been carried out in adult subjects with NAFLD or cryptogenic cirrhosis.     

Effects of dietary cholesterol and triglycerides on lipid concentrations in liver, plasma, and bile

Dietary cholesterol (CHL) and triglycerides (TG) can influence plasma, hepatic, and biliary lipid composition, but effects on lipids in these three compartments during the early stages of CHL gallstone formation have not been studied in parallel. We fed prairie dogs diets containing one of four tes oils (safflower, coconut, olive, or menhaden) at either 5 or 40% of calories, in the presence of 0 or 0.34% CHL, for 3 wk. In the absence of dietary CHL, increases in dietary TG produced 50–200% increases in the concentrations of biliary CHL and hepatic cholesteryl ester (CE), while the concentrations of hepatic free CHL (FC) as well as plasma FC and CE remained relatively unchanged. Increasing dietary CHL to 0.34% resulted in increases in hepatic FC of approximately 50% for all four fats regardless of whether they were supplied at 5 or 40% of calories. CHL supplementation caused more pronounced increases in biliary CHL (200–400%), hepatic CE (50–200%), plasma FC (up to 100%), and plasma CE (up to 150%), and these increases were exacerbated by concurrent supplementation of dietary fat and CHL (biliary CHL: 300–700%; hepatic CE: 100–250%; plasma FC: up to 165%; plasma CE: 100–350%). These results indicate that enhanced secretion of biliary CHL and, to a lesser extent, increased synthesis of hepatic CE, may be primary mechanisms for maintaining the hepatic FC pool. Furthermore, dietary CHL and high levels of fat intake are independent risk factors for increasing biliary CHL concentrations, and adverse effects on lipid concentrations in plasma and bile tend to be exacerbated by ingestion of diets rich in both fat and CHL.     

Theophylline-Based KMUP-1 Improves Steatohepatitis via MMP-9/IL-10 and Lipolysis via HSL/p-HSL in Obese Mice

KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) has been reported to cause hepatic fat loss. However, the action mechanisms of KMUP-1 in obesity-induced steatohepatitis remains unclear. This study elucidated the steatohepatitis via matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNFα), and related lipolysis via hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) by KMUP-1. KMUP-1 on steatohepatitis-associated HSL/p-HSL/ATGL/MMP-9/TNFα/interleukin-10 (IL-10) and infiltration of M1/M2 macrophages in obese mice were examined. KMUP-1 was administered by oral gavage from weeks 1–14 in high-fat diet (HFD)-supplemented C57BL/6J male mice (protection group) and from weeks 8–14, for 6 weeks, in HFD-induced obese mice (treatment group). Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining of tissues, oil globules number and size, infiltration and switching of M1/M2 macrophages were measured to determine the effects on livers. IL-10 and MMP-9 proteins were explored to determine the effects of KMUP-1 on M1/M2 macrophage polarization in HFD-induced steatohepatitis. Long-term administration of KMUP-1 reversed HFD-fed mice increased in body weight, sGOT/sGPT, triglyceride (TG) and glucose. Additionally, KMUP-1 decreased MMP-9 and reactive oxygen species (ROS), and increased HSL/p-HSL and IL-10 in HFD mice livers. In conclusion, KMUP-1, a phosphodiesterase inhibitor (PDEI), was shown to reduce lipid accumulation in liver tissues, suggesting that it could be able to prevent or treat steatohepatitis induced by HFD.     

Modeling Sialidosis with Neural Precursor Cells Derived from Patient-Derived Induced Pluripotent Stem Cells

Sialidosis, caused by a genetic deficiency of the lysosomal sialidase gene (NEU1), is a systemic disease involving various tissues and organs, including the nervous system. Understanding the neurological dysfunction and pathology associated with sialidosis remains a challenge, partially due to the lack of a human model system. In this study, we have generated two types of induced pluripotent stem cells (iPSCs) with sialidosis-specific NEU1^G227R and NEU1^V275A/R347Q mutations (sialidosis-iPSCs), and further differentiated them into neural precursor cells (iNPCs). Characterization of NEU1^G227R- and NEU1^V275A/R347Q- mutated iNPCs derived from sialidosis-iPSCs (sialidosis-iNPCs) validated that sialidosis-iNPCs faithfully recapitulate key disease-specific phenotypes, including reduced NEU1 activity and impaired lysosomal and autophagic function. In particular, these cells showed defective differentiation into oligodendrocytes and astrocytes, while their neuronal differentiation was not notably affected. Importantly, we found that the phenotypic defects of sialidosis-iNPCs, such as impaired differentiation capacity, could be effectively rescued by the induction of autophagy with rapamycin. Our results demonstrate the first use of a sialidosis-iNPC model with NEU1^G227R- and NEU1^V275A/R347Q- mutation(s) to study the neurological defects of sialidosis, particularly those related to a defective autophagy–lysosome pathway, and may help accelerate the development of new drugs and therapeutics to combat sialidosis and other LSDs.     

Endogenous lipolytic activities during autolysis of highly enriched hepatic lysosomes

Highly enriched (50- to 70-fold) fractions of “native” lysosomes were isolated using continuous flow electrophoresis from livers of rats which had not been pretreated with Triton WR-1339. Incubation of lysosomes for 30 min at pH 5.0 in the presence of 5 mM EDTA resulted in a dramatic loss in the content of fatty acids bound to triacylglycerols (137 down to 10 μmol/mg protein) and to phospholipids and an elevation in the level of unesterified fatty acid. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin concentrations decreased whereas those of lysophosphatidylethanolamine (0.8 up to 8.5% of total lipid-P) and lysophosphatidylcholine (1.9 up to 16.7%) rose in a manner parallel to their respective, fully acylated lipids. Other phospholipids, including phosphatidylinositol, did not change in concentration during incubation. These results indicate that lysosomal phospholipase A, sphingomyelin and triacylglycerol lipase are activated by incubation at acid pH, enabling them to hydrolyze endogenous lysosomal lipids. However, lysosomal phosphatidylinositol-directed phospholipase C is apparently unable to interact with phosphatidylinositol of the lysosomal membrane.     

Effect of magnesium deficiency on post-heparin lipase activity and tissue lipoprotein lipase in the rat

Previous studies have provided evidence that Mg deficiency affects lipid metabolism. The present experiments were designed to assess whether the hypertriglyceridemia associated with Mg deficiency was related to alterations in post-heparin lipase activity (PHLA). Mg-deficient and control diets were pair-fed to weanling Wistar rats for eight days and plasma lipoproteins were separated into various density classes by sequential preparative ultracentrifugation. Triglycerides were significantly increased in chylomicrons and in the very low density lipoprotein, low density lipoprotein and high density lipoprotein (HDL) fractions. Cholesterol and phospholipid levels were significantly lower in the HDL fraction. PHLA in deficient rat was substantially lower than in control rats. The inverse correlation between plasma triglyceride concentration and PHLA strongly suggests that hypertriglyceridemia is the result of defective lipolysis of plasma triglycerides in Mg-deficient rats. Further examination of the PHLA was carried out by salt-mediated inhibition of lipoprotein lipase (LPL) and by heparin sepharose affinity chromatography and purified rat LPL antiserum. The results indicate that hepatic lipase is significantly decreased in Mg-deficient rats but the low PHLA is due mainly to a decline in LPL. However, total LPL activity, that is, both the intracellular and the extracellular oools of LPL in adipose tissue, heart and diaphragm, were unaffected by Mg deficiency. The results suggest that the decrease of LPL activity in the plasma of Mg-deficient rats may be due to a selective decrease in the heparin-releasable pool of enzyme.     

Changes in serum lipids in rats treated with oral cooper

Disturbances in lipid metabolism during copper deficiency in rats are well recognized. Copper deficiency is associated with the spontaneous retention of hepatic iron. Previous studies have reported that hypercholesterolemia and hypertriglyceridemia are associated with elevated hepatic iron concentrations in copper deficient rats. There was a direct relationship between the magnitude of blood lipids and the concentration of hepatic iron. Based on these data, it has been hypothesized that iron was responsible for the development of lipemia of copper deficiency. In this study was determined the effect of increasing doses of Cu(10, 20 and 50 ppm) in the diet, on the serum total lipids, total cholesterol, triglycerides (triacylglicerols), phospholipids, non-esterified fatty acids (NEFA) and liver iron and zinc concentrations in normal rats. The results were compared with normal rats that received a balanced diet containing 0.6 and 6 ppm of Cu, respectively. The results show that Cu-supplement diminished the cholesterol and triglyceride serum levels, increased the level of phospholipids, NEFA and concomitantly decreased the hepatic concentrations of Fe and Zn. There was a statistically significant (p < 0.05) simple correlation between triglycerides and liver Fe (r = 0.917; R2 = 64.03%), cholesterol and liver Zn (r = 0.872; R2 = 76.07%), cholesterol and liver Fe (r = 0.995; R2 = 99.10%), liver Fe and liver Cu (r = -0.612), liver Fe and liver Zn (r = 0.837), liver Cu and liver Zn (r = -0.612), and serum triglycerides and liver Zn (r = 0.967). The mechanism(s) by which Fe and Zn determine these changes is not known; none of the enzymes that act in cholesterol and triglyceride metabolism and biosynthesis require Fe and/or Zn. The increase of NEFA is due to changes in the process of lipolysis and re-esterification of the fatty acids in blood. However, additional studies are needed for the precise mechanisms of this interrelationships to be clarified.     

Lipidomic Analyses of Female Mice Lacking Hepatic Lipase and Endothelial Lipase Indicate Selective Modulation of Plasma Lipid Species

Hepatic lipase (HL) and endothelial lipase (EL) share overlapping and complementary roles in lipoprotein metabolism. The deletion of HL and EL alleles in mice raises plasma total cholesterol and phospholipid concentrations. However, the influence of HL and EL in vivo on individual molecular species from each class of lipid is not known. We hypothesized that the loss of HL, EL, or both in vivo may affect select molecular species from each class of lipids. To test this hypothesis, we performed lipidomic analyses on plasma and livers from fasted female wild-type, HL-knockout, EL-knockout, and HL/EL-double knockout mice. Overall, the loss of HL, EL, or both resulted in minimal changes to hepatic lipids; however, select species of CE were surprisingly reduced in the livers of mice only lacking EL. The loss of HL, EL, or both reduced the plasma concentrations for select molecular species of triacylglycerol, diacylglycerol, and free fatty acid. On the other hand, the loss of HL, EL, or both raised the plasma concentrations for select molecular species of phosphatidylcholine, cholesteryl ester, diacylglycerol, sphingomyelin, ceramide, plasmanylcholine, and plasmenylcholine. The increased plasma concentration of select ether phospholipids was evident in the absence of EL, thus suggesting that EL might exhibit a phospholipase A₂ activity. Using recombinant EL, we showed that it could hydrolyse the artificial phospholipase A₂ substrate 4-nitro-3-(octanoyloxy)benzoic acid. In summary, our study shows for the first time the influence of HL and EL on individual molecular species of several classes of lipids in vivo using lipidomic methods.     

Acid Sphingomyelinase,a Lysosomal and Secretory Phospholipase C,Is Key for Cellular Phospholipid Catabolism

Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.     

Potential Antiviral Strategy Exploiting Dependence of SARS-CoV-2 Replication on Lysosome-Based Pathway

The recent novel coronavirus (SARS-CoV-2) disease (COVID-19) outbreak created a severe public health burden worldwide. Unfortunately, the SARS-CoV-2 variant is still spreading at an unprecedented speed in many countries and regions. There is still a lack of effective treatment for moderate and severe COVID-19 patients, due to a lack of understanding of the SARS-CoV-2 life cycle. Lysosomes, which act as “garbage disposals” for nearly all types of eukaryotic cells, were shown in numerous studies to support SARS-CoV-2 replication. Lysosome-associated pathways are required for virus entry and exit during replication. In this review, we summarize experimental evidence demonstrating a correlation between lysosomal function and SARS-CoV-2 replication, and the development of lysosomal perturbation drugs as anti-SARS-CoV-2 agents.     

Fatty acid distribution in the bovine pre- and postpartum testis

Testes from fetuses, calves, bulls and recently castrated animals were analyzed for total lipids, lecithin, cephalin, triglycerides, diglycerides, cholesteryl esters and cholesterol. Total lipids increase somewhat with age, but in the castrated animal the increase is more marked. Phospholipid content increases with age, but decreases in the castrated animal. Cholesterol decreases and triglyceride increases after birth and in the castrated animal. Polyunsaturated acids increase with age in all lipid classes. Eicosatrienoic acid is more abundant in fetal testicular lipids than in testes removed after birth. In the castrated testis there is a general decrease in the unsaturated fatty acids. Acids of the ω6 family are the predominant polyunsaturated acids and increase somewhat with age in all lipids. The ω3 family of polyunsaturated acids appears mostly toward the end of fetal life and increases after birth. Acids of the linoleate family reach approximately 25% of total acids in most lipid classes at maturity whereas the ω3 acids range from 1 to 9%. Presented at the American Dairy Science Association Meeting, Lexington, Ky., June 1965.     

Hormone-sensitive lipase: structure, function, evolution and overproduction in insect cells using the baculovirus expression system

Hormone-sensitive lipase (HSL) catalyses the rate-limiting stepin the hydrolysis of stored triacylglycerols and is therebya key enzyme in lipid metabolism and overall energy homeostasis.The gene organization of human HSL indicates that each putativefunctional region is encoded by a different exon, raising thepossibility that HSL is a mosaic protein. The catalytic serine(Ser423), as shown by site-directed mutagenesis, is encodedby exon 6. The phosphorylation site for cAMP-mediated activitycontrol and a second site, which is presumably phosphorylatedby 5' AMP-activated kinase, are encoded by exon 8, and a putativelipid-binding region is encoded by the ninth and last exon.Besides the catalytic site serine motif (GXSXG), found in virtuallyall Upases, a sequence similarity between the region surroundingthe catalytic site of HSL and that of five prokaryotic enzymeshas been found, but the functional basis of this is not yetunderstood. To resolve the 3-D structure of HSL, an expressionsystem utilizing recombinant baculovirus and insect cells hasbeen established. The expressed protein, 80 mg/l culture, hasbeen purified to homogeneity and a partial characterizationindicates that it has the same properties as HSL purified fromrat adipose tissue.     

Lung lipid synthesis from acetoacetate and glucose in developing rats in vitro

Acetoacetate (AcAc) and glucose were compared as energy sources and as precursors for lipid synthesis in the lungs of developing rats. Minced lung tissue was incubated with [3-14C]AcAc or [U-14C]glucose and the oxidation of each substrate to CO2 or its incorporation into tissue lipids was quantified. The highest rates of oxidation were obtained during the first 5 days for AcAc and the first 2 days of life for glucose and oxidation of AcAc was 3-4 times greater than that of glucose at all ages. Throughout postnatal development, the rates of nonsaponifiable lipid, fatty acid and hence total lipid (chloroform/methanol extractable) synthesis from AcAc were 2-3 times those of glucose. The highest rates of total lipid synthesis from AcAc and glucose were observed at birth. Glucose was utilized for glyceride-glycerol synthesis at a higher rate than AcAc. Similar patterns of incorporation of AcAc and glucose into various lipid classes were noted. Of the total lipids synthesized from AcAc and glucose, respectively, phospholipid plus monoglyceride accounted for 64% and 77%, triglyceride 13% and 13%, diglyceride plus cholesterol 11% and 4%, fatty acids 9% and 4%, and cholesteryl esters 3% and 1%. AT birth, the specific activities of all lipids except triglyceride derived from AcAc were greater than those from glucose. Rates of synthesis of all complex lipids declined with age. The results of these experiments demonstrate that AcAc is utilized more readily than glucose for energy production and lipid synthesis in developing rat lungs.     

Lipid class composition during embryonic and early larval development in Atlantic herring (Clupea harengus,L.)

The lipid class compositions of Atlantic herring eggs and larvae were determined immediately before fertilization, after fertilization and at various times during subsequent embryonic and early larval development. Total lipid constituted 15% of the dry wt of ripe eggs, 70% of the total lipid being polar lipid with phosphatidylcholine (PC) accounting for almost 90% of the polar lipid. In general, the total lipid content decreased gradually during embryogenesis and in particular during larval development. Within 3 hr after fertilization the relative percentage of neutral lipid decreased slightly. This was followed by a general decrease in polar lipid which, by the stage of yolk sac absorption, was reduced to 52% of the total lipid. The decreased percentage of polar lipid was due entirely to a decrease in PC, which was reduced to 66% of the polar lipids at the stage of yolk sac absorption. The accompanying increase in the percentage of neutral lipids was mainly due to increased percentages of triacylglycerols (TAG) up to yolk sac absorption and cholesterol esters in the larval stages. During the first 4 days after hatching, phospholipids and to a lesser extent cholesterol were preferentially depleted in the yolk sacs, which also had higher levels of free fatty acids. The results are discussed in relation to possible roles of different lipids during embryonic and early larval development.     

Relative utilization of fatty acids for synthesis of ketone bodies and complex lipids in the liver of developing rats

The regulation of hepatic ketogenesis, as related to the metabolism of fatty acids through oxidative and synthetic pathways, was studied in developing rats. [1-¹⁴C] palmitate was used as a substrate to determine the proportions of free fatty acids utilized for the production of ketone bodies, CO₂ and complex lipids. Similar developmental patterns of hepatic ketogenesis were obtained by measuring the production of either [¹⁴C]acetoacetate from exogenous [1-¹⁴C] palmitate or the sum of unlabeled acetoacetate and β-hydroxybutyrate from endogenous fatty acids. The production of total ketone bodies was low during the late fetal stage and at birth, but increased rapidly to a maximum value within 24 hr after birth. The maximal ketogenic capacity appeared to be maintained for the first 10 days of life.¹⁴CO₂ production from [1-¹⁴C] palmitate increased by two- to fourfold during the suckling period, from its initial low rate seen at birth. The capacity for synthesis of total complex lipids was low at birth and had increased by day 3 to a maximal value, which was comparable to that of adult fed rats. The high lipogenic capacity lasted throughout the remaining suckling period. When ketogenesis was inhibited by 4-pentenoic acid, the rate of synthesis of complex lipids did not increase despite an increase in unutilized fatty acids. During the mid-suckling period, approximately equal amounts of [1-¹⁴C] palmitate were utilized for the synthesis of ketone plus CO₂ and for complex lipid synthesis. By contrast, in adult fed rats, the incorporation of fatty acids into complex lipids was four times higher than that of ketone plus CO₂. These observations suggest that stimulated hepatic ketogenesis in suckling rats results from the rapid oxidation of fatty acids and consequent increased production of acetyl CoA, but not from impaired capacity for synthsis of complex lipids.