Lipid Inclusion Research Articles

Intracellular transport of cholesteryl esters from lysosomes to cytoplasm in macrophages.

The mechanism through which nonmembranous lipid inclusion bodies consisting of cholesteryl esters accumulate in the cytoplasm was studied. Most lipid inclusion bodies in macrophages after 24 h incubation with anisotropic cholesteryl oleate liquid crystals were surrounded by a limiting membrane. The limiting membrane, however, could not be observed after further incubation for 48 h in the presence of esterastin, which is known to be an inhibitor of lipase and esterase. Under these conditions, the levels of hydrolysis and re-esterification of cholesteryl esters were less than 15% and 5% of the control ones, respectively. These results suggest that the inclusion bodies were transferred from lysosomes to the cytoplasm, with partial hydrolysis of cholesteryl esters, in addition to through the pathway via microsomes.

Changes in individual rates of pancreatic enzyme and isoenzyme biosynthesis in the obese Zucker rat.

Both alterations of enzyme content and a markedly decreased secretory response to selected physiological stimuli have been demonstrated previously in the pancreas of the obese Zucker rat. The purpose of the present investigation was to determine the degree to which alterations of enzyme content could be attributed to changes in enzyme biosynthesis. Amylase content of obese rats was decreased by 50%, whereas lipase and trypsinogens were significantly increased. However, the decrease in amylase content was less than might have been predicted from the rate of amylase biosynthesis (80% decrease), and the increases in content of trypsinogen(s) and lipase were greater than would have been predicted from alterations in the absolute rates of biosynthesis. In view of the rapid turnover of pancreatic enzymes under normal conditions, it seems probable that a markedly decreased secretory response to various stimuli leads to an increased content of some enzymes in the pancreas of the obese rat. Ciglitazone treatment, which decreases insulin resistance in obese animals and leads to normalization of glucose metabolism in their pancreatic tissue, restored the enzyme-synthesis rates towards normal, showing that the abnormalities of enzyme synthesis were linked to the insulin resistance rather than to the obese genotype itself. Lipid inclusion bodies were found in acinar cells of obese rats. These bodies have previously been described in acinar cells of starved animals, which, in common with the acinar tissue of the obese Zucker rat, have decreased glucose metabolism.

Cholesteryl ester cycle in cultured hepatoma cells

The existence of a cholesteryl ester cycle in cultured Fu5AH hepatoma cells was documented and factors affecting the rate of turnover of the cholesteryl ester cycle in this cell line were explored. The influence of the physical state of the lipid inclusion in which the cholesteryl esters are stored could be addressed in this cell line because these cells can be induced to store cholesteryl esters in anisotropic (liquid-crystalline) cytoplasmic inclusions by exposure to free cholesterol-rich phospholipid dispersions or in isotropic (liquid) inclusions by addition of oleic acid to the phospholipid dispersions. To examine the relative rates of turnover of the cholesteryl ester cycle in the cells with the two types of inclusions, the fraction of cholesteryl linolenate, a cholesteryl ester present in low amounts in these inclusions, was examined after cells were exposed to medium containing linolenate. After 12 h, cells with anisotropic inclusions contained 17.5% cholesteryl linolenate and cells with isotropic inclusions contained 29.8% cholesteryl linolenate, suggesting an approximately 2-fold difference in turnover of the cholesteryl ester pool. To determine whether this difference was due to a differential rate of cholesteryl ester hydrolysis, the acyl CoA : cholesterol acyl transferase arm of the cholesteryl ester cycle was blocked using a specific inhibitor, Sandoz 58-035. In the presence of this compound, cholesteryl ester was hydrolysed twice as fast in cells with isotropic inclusions as compared to that in cells with anisotropic inclusions. The difference in rate of turnover of the cholesteryl ester cycle was shown to be related to the rate of hydrolysis of cholesteryl ester which, in turn, is related to the physical state of the stored cholesteryl ester.

急速凍結割断法による家兎動脈硬化壁細胞内脂質球の存在状態について

Rapidly-frozen, etched replica of atheromatous aorta of WHHL rabbit was examined. In foam cells, lipid droplets were seen in two sites; membrane-bounded droplets in lysosomes and cytoplasmic droplets without membranes surrounded by 10nm filaments. The latter droplets, which were frequently observed, showed concentric lamellar structure like an onion and may correspond to cholesterol ester-rich lipid inclusion isolated byLang& Insull (J. Clin. Invest. 1970) and us (Takano, T. et al. Biochem. Int. 1982).

Cholesterol ester hydrolase and sterol carrier proteins.

The cholesterol substrate required for sustained adrenal steroidogenesis is largely derived from the endogenous stores of cholesterol esters, which are located in large lipid inclusion droplets in the cytoplasm. In isolated adrenal cells, these esters are hydrolyzed during a variety of stimuli associated with cellular cAMP production. This largely appears to be a response to the action of a neutral cholesterol ester hydrolase, whose activity is modulated by phosphorylation of the enzyme protein, catalyzed by cAMP-dependent protein kinase. Transfer of the resulting unesterified cholesterol to mitochondria can be accomplished in a model system by sterol carrier protein2 (SCP2). This protein is distinct from fatty acid binding protein (FABP), has a Mr of 13,500 and is basic in nature. SCP2 can sequester cholesterol from lipid inclusion droplets in a stoichiometric relationship, and transfer this cholesterol to isolated adrenal mitochondria. SCP2 can also enhance the intermembrane transfer of mitochondrial cholesterol to cytochrome P 450scc, but does not directly affect cholesterol side chain cleavage. The stimulatory effect of adrenal cytosolic preparations on mitochondrial pregnenolone production can be completely abolished by pretreatment with anti SCP2 IgG.

Serological and histopathological responses of rainbow trout, Salmo gairdneri Richardson, to experimental infection with the 13P2 reovirus

Abstract. Both fingerling and adult rainbow trout were given intraperitoneal inoculations of the 131P2 reovirus which did not result in mortalities but produced a subclinical disease in 56% of the adults and in 65% of the juvenile fish. Sera from virus infected adult trout contained virus neutralizing and precipitating antibodies and demonstrated immunofluorescence of antigen(s) specific for the 13P2 virus. Virus infection was characterized by increased virus titres and a multifocal reticulo‐endothelial granulomatous hepatitis, occasionally accompanied by a fibrosing pancreatitis. Electron microscopy of infected liver tissues demonstrated arrays of 13P2 virus particles in a reticuloendothelial cell phagosome and in a hepatocyte lipid inclusion body from one of three juvenile fish examined. These results show that 13P2 virus inoculation of rainbow trout causes a non‐virulent virus infection accompanied by specific humoral antibodies. These antibodies are sufficient in titre for use in future serologic studies of the virus. Further discussion regarding the pathogenesis of this virus in bluegills versus rainbow trout is presented.

Polarizing microscopy of birefringent lipids in rabbit fetal lungs.

The development of birefringent lipids in pre- and postnatal rabbit lungs was studied by polarizing microscopy (PM) and correlated with transmission electron microscopy (TEM). Birefringent lipids first appeared as small scattered particles in the developing alveolar walls at 27 days of gestation. These particles became aggregated, gradually increased in amount in older fetuses, and reached a prenatal maximum at 30 days. Numerous particles were located in the alveolar lumen, beginning in the 30-day fetuses. Further increase in the amount of birefringent lipids occurred in the 60-day-old rabbits. TEM demonstrated cytoplasmic lipid inclusion bodies in the type II pneumocytes at 25 days of gestation. Some of these inclusions were lamellated, but regular lamellated bodies were often seen in older fetuses. It appears that the development of birefringent lipids is closely correlated with the development of lamellated bodies seen with TEM, and PM is a simple, useful method in following the development and maturation of surfactant lipids.

Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis.

The ability of sterol carrier protein2 (SCP2) to mediate transfer of unesterified cholesterol from adrenal lipid inclusion droplets to mitochondria has been tested in an in vitro model system. Unlike mitochondrial utilization of cholesterol added in acetone or dimethyl sulfoxide, the unesterified cholesterol of lipid droplets did not provide a readily available source of substrate for mitochondrial pregnenolone production, without the addition of a transport mediator. Addition of SCP2, but not albumin, stimulated mitochondrial utilization of droplet cholesterol in a concentration-dependent manner. In the absence of mitochondria, SCP2 sequestered lipid droplet cholesterol, and in the presence of mitochondria, which were unable to convert cholesterol to pregnenolone, this cholesterol was quantitatively accumulated by mitochondria. Both processes were concentration-dependent and demonstrated a molar ratio of SCP2 and cholesterol for both binding and transport of 1. SCP2 also enhanced pregnenolone formation by mitochondria which were incubated in the absence of an extramitochondrial source of cholesterol. However, SCP2 had no effect on steroid release from a crude particulate fraction. These studies suggest that the effects of SCP2 are related to delivery of cholesterol from preformed stores to and into mitochondria for initiation of steroid hormone synthesis, and may represent an important modulator of sterol metabolism in adrenal cortical cells.

The distribution of orthogonal arrays in the freeze-fractured rat median eminence.

The distribution of orthogonal arrays of particles and their relationships to gap and tight junctions have been studied in the glia of the freeze-fractured rat median eminence (ME). These rectilinear clusters of intramembrane particles are thought to represent trans-membrane channels for ions or metabolites, and were found to be densely packed on the membranous laminations of the pial-glial limitans. Additionally, arrays were found to be present on all of the perivascular glial end-feet examined. Two classes of end-feet were distinguished by their relative densities of orthogonal arrays. End-feet displaying low densities of arrays occurred more frequently in the internal zone, while end-feet displaying high densities occurred more often in the external zone. Similar distinctions based on array density could be made in membranes from other regions of the cell as well. Cross-fractures revealing the cytoplasm underlying these membranes often exposed lipid inclusion bodies, suggesting that membranes containing few arrays belong to tanycytes (or to 'astrocyte-like tanycytes'). The distribution of arrays appeared to be unrelated to the distribution of gap junctions in the membranes of astrocytes and tanycytes (and 'astrocyte-like tanycytes') of the ME, appearing near to and far from gap junctions with approximately equal frequency. Orthogonal arrays were absent from glial membranes near synaptic profiles in the ME. Arrays were also absent from the microvillous membranes of the apical surfaces of ependymal cells, from the cytoplasmic protrusions into the CSF of tanycytes, and from the vicinity of the tight and complex junctions linking the tanycyte and ependymal cell lateral membranes near their apical poles. These results suggest that there is a gradient of array density for most glia of the ME, increasing from the ventricular to the pial surface.

In vivo development of adipose tissue following implantation of lipid-depleted cultured adipocyte

The monolayer culture of isolated and disaggregated adipocytes from rat omental and perirenal sites, gave rise to a population of fibroblast-like cells, usually devoid of lipid inclusion. Similar fibroblast-like cells have been obtained in cultures of adipose tissue stromal cells and are thought to be undifferentiated adipocyte stem cells. Although the adipocyte-derived fibroblasts were morphologically indistinguishable from culture-derived fibroblasts of other origins, upon autoimplantation into the splenic bed they regained the lipid inclusion and developed again into adipose tissue. The findings suggest that the transformation of adipose cells into fibroblast-like cells is reversible modulation and not a dedifferentiation into the adipose tissue stem cell. This work also substantiates the increasingly recognized heterogeneity of fibroblasts.

Ultrastructural and histochemical identification of sclerosing liposarcoma.

A paratesticular tumour by light microscopy showed large amounts of partly hyalinized collagenous stroma, spindle shaped fibroblast-like cells, pleomorphic cells with foamy cytoplasm, multinucleated tumour type giant cells and lipid inclusions. By light microscopy, there was a problem as to whether the tumour should be classified with the liposarcomas or with the fibrous histiocytic group. Ultrastructurally, spindle shaped cells resembled fibroblasts and, in a few cells, intracellular collagen fibres were seen. Histochemical studies revealed the presence of mannose-rich glycoprotein which is characteristic for collagen producing cells, but frequently these cells also showed numerous lipid inclusion. The foamy cytoplasmic areas of pleomorphic cells were shown to consist of dilated ergastoplasmic sacs and large vacuoles lined by smooth endoplasmic reticulum negating the possibility that these cells were of histiocytic origin. This is supported further by the dearth of lysosomes and absence of lysozyme (muramidase) in the tumour cells. The presence of numerous lipid inclusions in many of the tumour cells, which are otherwise ultrastructurally similar to fibroblast, suggests that these cells represent precursors of lipoblasts. The above findings taken together allowed the tumour to be categorized as a sclerosing liposarcoma.

Relationship between the cholesterol esters and free cholesterol of individual atherosclerotic lesions and lesion size in aortas of rabbits on atherogenic and regression diets

A study was made on the cholesterol lipids and DNA in lesions of aortas of rabbits either fed continuously on a cholesterol-rich diet or fed the cholesterolrich diet for 6 weeks followed by a chow diet for up to 22 weeks. The DNA content of the aortic intimal—medial lesion samples was proportional to the surface area of these samples. The content of cholesteryl ester and unesterified cholesterol of the aortic lesions was compared with lesion area, lesion DNA and the topographical location on the thoracic and upper abdominal aorta luminal surface. The largest 80% of the lesions in an aorta contained a disproportionately large amount of cholesteryl ester compared to the smallest 20% of the lesions. The lesion content of free cholesterol compared to lesion area followed a nearly linear relationship. Similarly, cholesteryl ester content was non-linearly related to lesion DNA content but free cholesterol content was nearly proportionally related to DNA content. The non-linear relationships of cholesteryl ester content to lesion size and DNA content may be associated with a nonlinear relationship between lipid inclusion volume and lesion size. The linearity observed for free cholesterol content as a function of lesion size may be due to a linear relationship between lipid inclusion surface area and lesion size. Neither diet nor the topographical location of lesions appeared to be important factors in affecting the cholesterol lipid content of lesions. Lesions became more irregular in shape as they grew larger.

Cellularity of adipose tissue in fetal pig.

Adipose tissue cellularity was studied in the 85-day-old Large-White pig fetus. The aim of this work was to count the adipose cells of forming tissue in an animal species which could be a possible model for studying adipose tissue in humans. Using a morphometric method with electron microscopy, mean triglyceride volume per cell was determined independently of mean cell volume. This method is suitable for counting adipose cells in the early stage of differentiation whatever their size and lipid inclusion volume. Site-by-site dissection of adipose tissue was not feasible in the 85-day old fetus and adipose cell number was computed by dividing total carcass triglyceride volume by mean triglyceride volume per cell. The carcass triglyceride seemed to originate only from adipose cells. The mean total carcass triglyceride volume per fetus (1.84 g) was low but, owing to the low mean triglyceride volume per cell (180.28 microns3), the adipose cell number (11.15 X 10(9)) was relatively important, as it represented about 27% of the extramuscular adipose cell number in the Large-White adult pig (41 X 10(9)).

粥状硬化の成因に関する微細構造的研究 (第1報)

Ten atheromatous rabbits (1% cholesterol feeding, for 12-14 weeks), weighting 3.2-3.8kg were used. All rabbits were injected intrarenously with 5ml/Kg of 50% carbon suspension. Aortic specimens of 5 rabbits were taken 24hrs. after the injection of carbon suspensions, but other 5 aortic specimens were taken 7 days after the injection.The cytoplasm of endothelial cells contained numerous lipid inclusions and carbon particles. These lipid inclusions and carbon particles were partially surrounded by the single electrondense layers. There were no carbon particles in lipid inclusions.Typical foam cells contained lipid particles, carbon particles, ceroid bodies and multilaminated membrane structures (myelin fogures). Ceroid bodies were composed of numerous lipid droplets, laminated vesicles and granules.The carbon particles were frequently found in ceroid bodies or myelin figures, but there were no carbon particles in lipid inclusions.The smooth muscle cell contained numerous lipid inclusions soursounded by electron-dense layers (deposits).It was suggested that foam cells in the subendothelial layer varied according to their stage of development, especially a lipid inclusion (particle)/ceroid body ratio.Multilayered vesicles, granules and lipid droplets which were derived from the destoroyed cytoplasm of foam cells were frequently observed in extracellular spaces.

Intranuclear Inclusion Bodies in Epithelial Cells of Human Vas Deferens

Four different types of inclusion bodies were seen within the nucleus of the principal cells in segments of vas deferens from 40 fertile males: (1) electron-dense homogeneous bodies of various sizes (0.1--1.8 microns) and shapes, (2) granular bodies approximately 0.06--0.12 microns, (3) lipid inclusion bodies, and (4) less electron-dense, filamentous bodies. They were either membrane-bound or free within the nucleoplasm. Some membranous material was scattered within the nucleoplasm. The internal structure of nuclei containing these bodies differed from cell to cell. The nucleoplasm was composed either of dense granular elements or fine filamentous components. The aggregation and density of the ground nuclear material increased with increasing numbers of inclusion bodies. Very dense nuclei were in cells with typical features of degeneration. Frequently, extruded parts of cells or entire cells were seen within the lumen of the vas deferens.

Quantitative ultrastructural investigations of mitochondrial development in Leishmania donovani during transformation.

The ultrastructure of the mitochondrion during transformation of Leishmania donovani from amastigote to promastigote stages was studied by morphometric analysis. There was a slight but significant decrease in relative mitochondrial volume (Vvmi) during transformation. This decrease was linear with time for at least 27 hr. No change in relative lipid inclusion volume (Vvli) was observed during transformation. The ratio of inner to outer mitochondrial surface membrane densities (Svmii:Svmio) also remained unchanged. Although the relative number of cristae remains unchanged after transformation, there is an increase in cristae number in the portion of the kinetoplast opposite the flagellum.

Experimental autoimmune adrenalitis in the rat

A single innoculation of an autologous adrenal extract produced autoimmune adrenalitis in rats. The histological changes were correlated with the plasma corticosterone levels and the capacity of in vitro steroid biosynthesis. Peripheral corticosterone values were determined by the competitive protein binding method. The in vitro studies were performed using [ 14C]-progesterone as precursor steroid and incubating with glandular tissue. The immunized female rats showed a significant decrease in plasma circulating corticosterone: 18 ± 3.6 μg 100 ml to 13.5 ± 1.4 μg/100 ml ( P < 0.05). In vitro studies demonstrated a lower conversion into corticosterone in incubations with the tissue of animals with adrenalitis, possibly due to two facts with a parallel action: (1) Alteration in the 11-betahydroxylase system and in the enzymatic cofactors responsible for the hydroxylation of desoxycorticosterone in C 11. Electron microscopy revealed partially destroyed mitochondrial cristae and lipid inclusion bodies, thus confirming this biochemical fact; (2) Slow metabolization of the precursor steroid added to the incubation medium, consequent upon a more accelerated exhaustion of the glandular tissue in immunized animals. The conclusion arrived at is that immunization provokes tissue lesions in the adrenal glands of female rats discernible both by light and electron microscopy. These lesions can be correlated with the biochemical findings in so far as concerns steroid synthesis.

Histochemical observations on the juxtanuclear complex of organelles in the hamster oocyte

The hamster ovarian oocyte surrounded by 2-3 layers of granulosa cells develops a large juxtanuclear complex of organelles, consisting of basophilic substance, mitochondria and some lipid inclusion bodies. The basophilic substance staining for RNA, protein and some diffuse lipoprotein forms the material with which the granular mitochondria, composed of protein and phospholipid, apparently multiply. The lipid inclusion bodies consist of phospholipids. The results of this histochemical investigation have been correlated with those of ultrastructural studies on the hamster oocyte. The paranuclear complex of organelles described in this paper has also been compared and contrasted with that seen in the primordial oocyte of hamster, as reported in the previous publication. The functional significance of juxtanuclear entities has been discussed in relation to the multiplication of organelles to be accumulated during oocyte growth.

Unusual lysosomes in hamster hepatocytes

Residual bodies (lysosomes) in hepatocytes of the Syrian golden hamster possess a relatively large electron-lucent lipid inclusion. In males on normal chow the lipid inclusions are large enough, in periportal hepatocytes, to be visible by light microscopy when frozen sections are stained by Oil red O. Thus, the pericanalicular arrangement of the lysosomes may be revealed either by Oil red O staining or by incubation for acid phosphatase activity. Demonstrable acid phosphatase activity is restricted to the area surrounding the lipid inclusion and, usually, to a region at one pole of the lysosome that contains electron-opaque materials characteristic of residual bodies generally. Feeding either female or male hamsters cholesterol-supplemented chow results in a dramatic increase in the number of lipid-containing lysosomes visible by light microscopy. Chemical determinations demonstrate a rapid and sustained rise in liver cholesterol levels, and the presence of cholesterol in lysosomes is shown by electron microscopy of tissue fixed with digitonin-containing aldehydes. Polarizing microscopy reveals the pericanalicular arrangement of anisotropic lipid. Electron microscopic images suggest that the lipid inclusion forms in GERL or neighboring ER. Discontinuing the cholesterol diet leads to a gradual disappearance of the electron-lucent lipid; residual bodies now resemble lipofuscin granules. It is suggested that hamster liver may serve as a model for the study of inherited disorders of lipid metabolism such as Wolman's disease and cholesteryl ester storage diseases, and other diseases in which lysosomes are filled with electron-lucent lipid.

An electron microscopic study of macroglia and microglia in the lateral funiculus of the developing spinal cord in the fetal monkey.

An electron-microscopic study has been made of the glial cells in the developing lateral funiculus of the cervical spinal cord in fetal rhesus monkeys. The various macroglial cell types, their precursor cells, and microglia are discussed in detail. An astrocytic lineage is proposed in which glioblasts present in the lateral funiculus give rise to astroblasts that then develop into mature astrocytes. Oligoblasts apparently migrate into the lateral funiculus as such and develop into active oligocytes. The active oligocytes become most predominant during the initial stages of myelinogenesis and are in direct continuity with developing myelin. The active oligocytes develop into mature oligocytes after myelination is completed. Microglia cells are present throughout development as three forms; resting microglia, globose microglia, and active microglia. The globose and active microglia predominates at specific times early in development when degeneration of apparent neuronal processes is taking place. The microglia cells are characterized by dense nuclear chromatin clumps, lipid inclusion bodies, dense vesicles, and, often, intracellular debris.