Autoradiographic localization of vitamin A in the adrenal gland of rats.

Cellular distribution of vitamin A in the rat adrenal was evaluated by autoradiography. Vitamin A was concentrated in the lipid droplets of epithelial cells of the zona fasciculata and zona reticularis. A small amount of vitamin A was also present in the cytoplasm and nucleus of these cells. The zona glomerulosa contained very little vitamin A either in lipid droplets or in the remainder of the cell. The medulla had essentially no vitamin A. Such strong cellular specificity for uptake of vitamin A by the cells of the zona fasciculata and zona reticularis supports the hypothesis that vitamin A is involved in synthesis of glucocorticoid hormones.     

Effects of melatonin on the ultrastructure of the golden hamster parathyroid gland.

Ultrastructural changes of the parathyroid glands of melatonin-treated golden hamsters were studied. Many chief cells in the parathyroid glands after 1 hour of administration of melatonin contained poorly-developed Golgi complexes associated with a few prosecretory granules and numerous lipid droplets as compared with those of the control animals. The morphology of the parathyroid glands after 5 hours of administration resembled that of the control animals. Many chief cells in the parathyroid glands after 24 hours of administration had well-developed Golgi complexes and cisternae of the granular endoplasmic reticulum, numerous prosecretory granules, a few lipid droplets and many secretory granules in the peripheral cytoplasm as compared with those of the control animals. The ultrastructure of the parathyroid glands after 48 hours of administration was almost similar to that of the control animals. It is considered that melatonin affects the secretory activity of the parathyroid gland.     

Alterations within the rat thyroid gland during vitamin A deficiency

Thyroid glands from female rats kept vitamin A deficient for one, two, and three months were examined by electron microscopy. After one month on the diet, no consistent alterations were noted. After two months, the colloid in some follicles displayed a peripheral zone of decreased density. In addition, ultimobranchial follicles within the gland had become keratinized. After two to three months on the diet, cells were seen entering the colloid. Many of these cells were identified as follicular cells since they often occurred in groups and occasionally exhibited remnants of desmosomes. Often the cells within the colloid appeared vacuolated, and by light microscopy were thought to contain lipid. However, electron microscopy revealed that these cells contained many digestive vacuoles rather than lipid droplets. Quantitative and autoradiographic studies indicated that thyroids of vitamin A deficient rats took up less radioiodide than thyroids of control rats. The keratinization of ultimorbranchial follicles in vitamin-A deficiency has been suggested as preliminary in the histogenesis of squamous cell carcinoma. However, an effect of vitamin A deficiency on thyroid follicular cells has not heretofore been reported. It's possible that the presence of follicular cells in the colloid reflects an accelerated turnover of these cells and could indicate an early pathological sign.     

Effects of dietary retinoid and triglyceride on the lipid composition of rat liver stellate cells and stellate cell lipid droplets

Hepatic stellate cells store the majority of the liver's retinoid (vitamin A) reserves as retinyl esters in stellate cell lipid droplets. A study was conducted to explore the effects of differences in dietary retinoid and triglyceride intake on the composition of the stellate cell lipid droplets. Weanling rats were placed on one of five diets that differed in retinoid or triglyceride contents. The dietary groups were: 1) control (2.4 mg retinol (as retinyl acetate)/kg diet and 20.5% of the calories supplied by triglyceride (as peanut oil]; 2) low retinol (0.6 mg retinol/kg diet and control triglyceride levels); 3) high retinol (24 mg retinol/kg diet and control triglyceride levels); 4) low triglyceride (2.4 mg retinol/kg diet and 5% of the calories supplied by triglyceride); and 5) high triglyceride (2.4 mg retinol/kg diet and 45% of the calories supplied by triglyceride). Stellate cells were isolated using the pronase-collagenase method and stellate cell lipid droplets were isolated by differential centrifugation. The levels of retinoids and other lipids were measured by high performance liquid chromatography. The stellate cells from control rats contained 113 micrograms total lipid/10(6) cells. Control stellate cell lipid droplets had the following mean percent lipid composition: 39.5% retinyl ester; 31.7% triglyceride; 15.4% cholesteryl ester; 4.7% cholesterol; 6.3% phospholipids; and 2.4% free fatty acids. Both the concentration of stellate cell lipids and the composition of stellate cell lipid droplets were markedly altered by changes in dietary retinoid. The low and high retinol groups contained, respectively, 82 and 566 micrograms total lipid/10(6) cells, with retinyl ester representing, respectively, 13.6% and 65.4% of the lipid present in the stellate cell lipid droplets. Low and high triglyceride groups were similar to controls in both stellate cell lipid content and the composition of the stellate cell lipid droplets. These findings indicate that the composition of stellate cell lipid droplets is strongly regulated by dietary retinoid status but not by dietary triglyceride intake.     

Retinyl ester storage particles (retinosomes) from the retinal pigmented epithelium resemble lipid droplets in other tissues

Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored.     

Collagen production by rat liver fat-storing cells in primary culture

Morphological changes, proliferation and collagen synthesis of fat-storing cells (FSC) in primary culture were examined. FSC, isolated from rats treated with vitamin A, showed numerous large lipid droplets in the cytoplasm and positive desmin staining. After 4-7 days culture, these cells were transformed into fibroblast-like cells with a gradual depletion of lipid droplets and with abundant well-developed rough endoplasmic reticulum. The proliferation analysis revealed that DNA synthesis preceded the increase of cell number. Enhancement of the collagen synthesis by FSC were associated with the morphological change of the cells. Quantitative analysis revealed that these cells produced mainly type I collagen (84%) and a small amount of type III collagen (16%).     

Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.     

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.     

Isolation and characterization of vitamin-A-storing lung cells

Vitamin A-storing cells have been shown to be distributed among various organs and tissues, including the lungs. In order to investigate this unique type of cell, the in vitro isolation has been carried out from rat lungs. Lungs were perfused with EGTA and collagenase solution in situ, and were digested with trypsin-collagenase solution at 60-min intervals for 2 h. Then, the cell suspensions obtained were incubated at 37 degrees C in F-10 medium supplemented with 10% fetal bovine serum (FBS) for 72 h. Non-adherent cells were then removed by vigorous washing with medium, and the resultant cell monolayer was harvested with trypsin to remove the contaminating macrophages. These cell fractions were shown to contain more than 96% of vitamin A-storing cells, judged by electron and fluorescence microscopic examinations. The cells grown in vitro retained well the overall morphology characteristic of the vitamin A-storing cells found in lung tissues. The isolated cells grew well in vitro and the growth was inhibited by D-valine or cis-hydroxyproline. The progeny of the cells still contained vitamin A lipid droplets after several transfer generations. Characteristic networks of fibronectin were also demonstrated around the cells. These results have shown that vitamin A-storing cells in the lung was successfully isolated from rat lungs and the cells possessed fibroblast-like characters storing vitamin A in small lipid droplets.     

Peroxisomes of the midgut epithelium, malpighian tubules and fat body of larvae of the dragonfly, Aeshna cyanea

The enterocytes of the midgut epithelium of Aeshna cyanea larvae are rich in peroxisomes while the nidal regenerative and endocrine cells contain only a few. Most of the enterocytic peroxisomes are microperoxisomes lacking a crystalloid nucleoid, but peroxisomes with well developed nucleoid are also present. The peroxisomes are usually concentrated in the basal region of the cells but may also spread into the apical region and closely intermingle with absorptive lipid droplets. They significantly increase in number, when the larvae are regularly fed lipid-rich natural food or long-chain monounsaturated fatty acids that are unusual dietary components of these animals. This observation seems to indicate that the enterocytic peroxisomes are involved in chain shortening and degradation of fatty acids absorbed from the gut lumen. Numerous microperoxisomes are also present in the lipid-storing cells of the Malpighian tubules and fat body.     

Chemical characterization of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth,Bombyx mori

Accumulation of lipid droplets within the cytoplasm is a common feature of the pheromone gland cells of many lepidopteran species. The cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori, were effectively extracted by dipping the trimmed glands in acetone for 10 min. In order to analyze the components originating from the lipid droplets, we separated the acetone extracts prepared before and after adult eclosion using HPLC, and specified the peaks showing a similar pattern of stage-dependence to that in the morphological change of the lipid droplets previously reported by Fónagy et al. (Arthropod Struct. Dev. 30 (2001) 113). Finally, we specified the peaks #1-5 and #1a-4a separated by reversed-phase HPLC as lipid droplet contents. Structure elucidation using FAB-MS and MS-MS analyses confirmed that they were triacylglycerols (TGs), and 12 species of TGs were identified as lipid droplet contents. Fatty acyl groups contained in these TGs were limited to five unsaturated C16 and C18 fatty acyl groups (delta 11-hexadecenoate, delta 10,12-hexadecadienoate, delta 9-octadecenoate, delta 9,12-ocatadecadienoate, and delta 9,12,15-ocatadecatrienoate), including the pheromone precursor delta 10,12-hexadecadienoate as a major component. Digestion with porcine pancreatic lipase confirmed that three major TGs eluted in the peaks #3-5 all contained C18 fatty acyl groups at the sn-2 position, indicating that the pheromone precursor is sequestered preferentially at the sn-1 and/or sn-3 position. Present results combined with the fact that the morphological change of the lipid droplets is under the control of PBAN indicate that the role of the cytoplasmic lipid droplets in the pheromone-producing cells is to store the pheromone precursor in the form of TGs and to provide it for pheromone production in response to the external signal of PBAN.     

A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

Ultrastructural evidence for estradiol synthesis in the ovary of persistent-estrous rats exposed to continuous illumination

The relation between sex hormone levels in blood and ultrastructural changes of ovarian follicles was examined in persistent-estrous rats exposed to continuous illumination (LL) for 100 days. Plasma LH showed a tonic level secretory pattern, and circulating estradiol and estrone concentrations were relatively high, while both levels of FSH and progesterone were low. Various stages of growing and degenerating follicles were observed in the ovary of the LL-exposed rats. The early stage of antral follicle did not seem to possess the ability of steroidogenesis. Theca cells around mature antral follicles contained prominent Golgi apparatuses, plenty of smooth endoplasmic reticulum (ER), abundant free ribosomes and many round-mitochondria. A few newly formed lipid droplets were seen in some of theca cells. Granulosa cells contained much distended rough ER, well-developed mitochondria, several lipid droplets and microfilaments. The theca cells of abnormal follicles with hyperplastic and infolded layers of granulosa cells contained many lipid droplets. However, the development of the smooth ER became hindered with increasing lipid droplets in the theca cell. On the other hand, granulosa cells of abnormal follicles contained greater numbers of lipid droplets than those of antral mature follicles, and were equipped with well-developed cytoplasmic organelles as were those of mature antral follicles. Theca interna cells of abnormal follicles may be more involved in the secretion of androgen, which has already been accumulated in the lipid droplets, than the cells involved in the active synthesis of the hormone, while the granulosa cells may convert its androgen to estrogen. The present findings suggest that both follicles of mature and abnormal types in the LL-exposed rat retain enough capacity of estradiol production and participate in the continued elevation of circulating estradiol, probably resulting in the stimulation of the theca cells by the tonic level of LH and of the granulosa cells by the levels of FSH, which are lower than the basal values during the normal 4-day estrous cycle.     

Localization of vitamin A in the small intestine of mouse

Summary Localization of vitamin A in the small intestine of mice was studied with electron microscope radioautography after administration of tritiated vitamin A. The label was concentrated over lipid droplets in cells distributed in the lamina propria and the submucous layer. The cells were similar both to fibroblasts and to fat-storing cells in their morphological features. The name Vitamin A-Storing Cell is proposed for these labeled cells, including the fat-storing cell in the liver.     

Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation

Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A-phosphorylated HSL and perilipin A.     

Fine structure and cytochemistry of lysosomes in the Ito cells of the rat liver

Summary Ultracytochemical studies of the performic acid-phosphotungstic acid (PFP) reaction and acid phosphatase (ACPase) activity in the Ito cells (fat-storing cells) of the rat liver revealed two kinds of lipid droplets: one surrounded by a structure giving PFP- and ACPase-positive reactions, recognized as a lysosome, the other without such a reactive structure displaying a limiting membrane.To elucidate the function of the lysosomes surrounding lipid droplets, experiments were carried out on the following groups of animals: (1) Vitamin A-deficient rats were fed a normal diet containing vitamin A, and (2) hypervitaminosis A was experimentally induced in previously untreated rats. Lipid droplets were studied in both groups.No lipid droplets reappearing in an early stage after restoration of the regular diet were either membrane-bounded or surrounded by lysosomes. Lipid droplets surrounded by lysosomes could be seen in rats fully restored from vitamin-A deficiency and more frequently in animals suffering from hypervitaminosis A. It seems likely that as a result of the lysosomal activity in the immediate vicinity of the lipid droplets a degradation of the vitamin A-containing lipid droplets takes place in the Ito cells. Therefore, the lysosome-surrounded lipid droplets can be regarded as a sort of autophagolysosome; these lysosomes may play a role in preventing an unrestricted increase in the number and volume of lipid droplets.This work was supported by Grants-in-Aid for Co-operative Research (Nos. 437001 and 57370001) from the Ministry of Education, Science and Culture, Japanese Government     

Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters

In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.     

Effects of hypergravity environment on the parathyroid gland of the isoproterenol-treated hamster

The ultrastructure of the parathyroid glands of hamsters subjected to 5 g environment after an intraperitoneal injection of isoproterenol was studied. In the isoproterenol-treated hamsters exposed to hypergravity environment, the Golgi complexes, cisternae of the granular endoplasmic reticulum and lipid droplets were significantly increased and secretory granules were significantly decreased compared with those of the control group. In addition, many chief cells contained numerous prosecretory granules in the Golgi areas and several secretory granules were situated close to the plasma membrane of the chief cells. These results suggest that the synthesis and release of parathyroid hormone may be markedly stimulated in the parathyroid glands of the isoproterenol-treated hamsters exposed to hypergravity environment.     

Relationship between mitochondria and the development of specific lipid droplets in capillary endothelial cells of the respiratory tract in patients with sarcoidosis

The aim of this study was to morphologically evaluate damage in the capillary endothelial cells of the respiratory tract in patients with sarcoidosis. We examined tissues of the bronchus and lung obtained from 16 patients with sarcoidosis consisting of 2 stage 0, 10 stage I and 4 stage II patients, and 11 controls. The morphology of capillary endothelial cells was studied using electron microscopy. In the samples from patients with sarcoidosis, lipid droplets exhibiting dark monophasic density (unsaturated fatty acids) were surrounded by abundant lysosomes in the capillary endothelial cells, and the double-membrane structure of the mitochondria attached to these lipid droplets was lost in three cases. Biphasic lipid droplets with dark and lucent (saturated fatty acids) densities were also observed, accompanied by a few lysosomes containing the residual bodies of undigested lipid-containing substances. Lucent monophasic droplets were also detected in the tissues from patients with sarcoidosis. The plasma membrane was more often damaged in capillary endothelial cells containing biphasic lipid droplets, lucent monophasic droplets as well as in those with dark monophasic droplets. However, no lipid droplets were detected in capillary endothelial cells obtained from the control subjects, except in a single case. This study demonstrated that a large number of mitochondria were mobilized and showed notable morphological changes including swelling in the capillary endothelial cells in patients with sarcoidosis. A close relationship between mitochondria and lipid droplets was observed in capillary endothelial cells of the respiratory tract, and this relation may be involved in the pathogenesis of sarcoidosis.