LIPOFUSCIN (AGING) PIGMENT GRANULES OF THE NEWBORN HUMAN LIVER

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and β-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.     

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.     

Ultrastructure of testicular macrophages in aging mice

Testicular macrophages of aging mice were studied by TEM. Testicular macrophages retained with Leydig cells the close morphological relationships observed in the adult young animals, but digitations were not found. Lipofuscin granules like those of the Leydig cells from aging mice were observed in the cytoplasm. These organelles were generally absent in the testicular macrophages of young adult mice. Testicular macrophages did not display phagocytosis of the lipofuscin granules. In addition, the latter were not found in the intercellular spaces. These observations indicated that lipofuscin granules were formed, at least in a great part, within testicular macrophages as a consequence of metabolic changes occurring with age. Fine lamellar organization was seen in the lipofuscin granules of both Leydig cells and testicular macrophages. Frequently, lipofuscin granules originated from secondary lysosomes containing lipidic vacuoles only. Together with accumulation of the lipofuscin granules, changes of testicular macrophage fine morphology were observed. Endoplasmic reticulum and Golgi apparatus became poorly developed, and coated vesicles were rarely found. Fewer mitochondria were encountered, but their ultrastructure was not altered. These results suggest that in testicular macrophages lipofuscin accumulation is associated with a functional involution.     

Lipofuscin-like fluorophores originated from malondialdehyde

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of post-mitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. In the present mini-review, I provide a comprehensive summary of fluorescent adducts originated from malondialdehyde (MDA).     

Effects of prolonged ACTH-stimulation on adrenocortical accumulation of lipofuscin granules in aged rats

Subcellular deposition of lipofuscin granules is a marker of aging. Human and rodent adrenal cortices accumulate lipofuscin granules with age, but the mechanism that leads to the accumulation is not known. The ultrastructural appearance of lipofuscin granules resembles that of secondary lysosomes. Since adrenocortical subcellular events are predominantly influenced by ACTH action, we therefore studied the effect of prolonged ACTH-stimulation on adrenocortical accumulation of secondary lysosome-like granules, designated herein as lipofuscin granules. Using aged Fischer 344 male rats as a model, we found that a 7 day ACTH stimulation exerts a reducing effect on adrenocortical lipofuscin accumulation. Thus, adrenocortical accumulation of lipofuscin granules with age in vivo may not be an irreversible process.     

Secretion of immunoreactive ANF from FRTL-5 rat thyroid cells: regulation by calcium ionophore A23187.

Atrial natriuretic factor-like immunoreactivity (ir-ANF) was characterized in a continuous line of rat thyroid follicular cells (FRTL-5) and the influence of the calcium ionophore A23187 on ir-ANF secretion was examined. Ir-ANF was identified by immunohistochemical staining as primarily reticular and juxtanuclear in short-term cultures, and more peripheral and granular in longer-term cultures, suggesting a process of ir-ANF packaging into secretory granules. The accumulation of ir-ANF granules was dependent upon the presence of thyrotropin (TSH) in the medium. Secreted ir-ANF was characterized using reversed-phase, high-performance liquid chromatography (RP-HPLC) and radioimmunoassay as a single peak eluting one fraction earlier than 125I-labeled rat ANF (99-126) (i.e., circulating atrial ANF) included as an internal standard. A23187 treatment of cells exhibiting primarily reticular ir-ANF caused a change to a pattern of more distinct, peripherally localized granules. This change occurred within 1 h after A23187 treatment and was dependent on the presence of Ca2+ in the medium. In cultures containing primarily ir-ANF granules, A23187 (0.5 micrograms/ml) induced a peripheral translocation of the granules at 30 min and a complete degranulation by 7 h. Enzyme-linked immunoadsorbent assay (EIA) confirmed a dose-dependent effect of A23187 on ir-ANF release into the medium. These results suggest that some of the effects of Ca2+ in the thyroid could be ascribed to its mobilization and release of ir-ANF, which in turn may have autocrine effects on thyroid follicular cells.     

Cytochemical and Histochemical Aspects of the Digestive Gland Cells of the Mussel Mytilus galloprovincialis (L.) in Relation to Function

The epithelium of the digestive tubules of the mussel Mytilus galloprovincialis is comprised of two cell types, namely digestive and basophilic cells. In basophilic cells, the secretory granules are beta-glucuronidase immunoreactive, a fact that enhances the hypothesis that beta-glucuronidase is synthesized in basophilic cells. A novel observation at the ultrastructural level is the pinocytic activity associated with the formation of coated pits. This observation constitutes direct evidence for endocytic processes taking place in basophilic cells. The use of cryostat sections from the same digestive tubules reveals, in many instances, a very pronounced neutral lipid accumulation in the same structures giving a positive reaction for N-acetyl-beta-hexosaminidase, indicating the association of those lipids with lysosomes. In some mussels, a high content of lipofuscin was observed in the lysosomes of the digestive cells. In these cases, the lysosomal structures show a limited neutral lipid content, and a weaker N-acetyl-beta-hexosaminidase reaction. In the digestive cells, the carbohydrate content of the lysosomes, and very well-developed canal system in the apical part of cells are discussed in relation to their function.     

Age-related differences in oxidative protein-damage in young and senescent fibroblasts

Aging is accompanied by an accumulation of oxidized proteins and cross-linked modified protein material. The intracellular formation and accumulation of highly oxidized and cross-linked proteins, the so-called lipofuscin, is a typical sign of senescence. However, little is known whether the lipofuscin accumulation during aging is related to environmental conditions, as oxidative stress, and whether the accumulation of oxidized proteins and lipofuscin is preferentially taking place in the cytosol or the nucleus and finally, what is the role of lysosomes in this process.Therefore, we investigated human skin fibroblasts in an early stage of proliferation (“young cells”) and in a late stage (“senescent cells”). Such cells were compared for the amount of protein carbonyls and lipofuscin and their distribution within the cytosol and the nucleus. Furthermore, cells were exposed to single and repeated doses of hydrogen peroxide and paraquat, measuring the same set of parameters. In addition to that the role of the proteasome to degrade oxidized proteins in young and senescent cells was tested. Furthermore, detailed microscopic analysis was performed testing the intracellular distribution of lipofuscin. The results clearly demonstrated that repeated/chronic oxidative stress induces a senescence-like phenotype of the distribution of oxidized proteins as well as of lipofuscin. It could be demonstrated that most of the lipofuscin is located in lysosomes and that senescent cells contain less lysosomes not lipofuscin-laden in comparison to young cells.     

Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of postmitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. In the present study, we raised a monoclonal antibody (MAb 1F83) directed to the malondialdehyde-modified protein and identified a lipofuscin-like dihydropyridine fluorophore as the major epitope. This antibody was used to conclusively demonstrate that the fluorophore forms on oxidatively modified low density lipoproteins. In addition, we demonstrated that the materials immunoreactive to MAb 1F83 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between the lipid peroxidation-derived aldehyde and primary amino groups of protein might represent a process common to the formation of the lipofuscin-like fluorophore during aging and its related diseases.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

CHANGES IN FINE STRUCTURE AND ACID PHOSPHATASE LOCALIZATION IN RAT THYROID CELLS FOLLOWING THYROTROPIN ADMINISTRATION

Shortly after the administration of 1/40 unit thyrotropin to rats, 24 hours post-hypophysectomy, the following sequence of changes has been observed within thyroid follicular epithelial cells: (1) the appearance of apical cell surface activity consisting of pseudopods projecting into the follicular lumen; (2) apparent phagocytic engulfment of colloid droplets lacking indications of acid phosphatase activity; (3) close association and probable fusion of newly formed colloid droplets and dense granules, the latter cytochemically positive for acid phosphatase activity; (4) the appearance of presumptive acid phosphatase activity within colloid droplets; and, (5) further colloid droplet changes, viz., basipetal migration and decrease in size, accompanied by an increase in density and in demonstrable acid phosphatase activity. These changes appeared to represent the resorption and degradation of follicular colloid. Comparable results were obtained using intact and more heavily stimulated animals. Colloid biosynthesis was tentatively visualized in these cells as a separate mechanism involving small vesicles prominent in the Golgi region and beneath the apical plasma membrane of some, but not all, thyroid follicular cells in each specimen.     

Accumulation of mercury in the anterior pituitary of rats following oral or intraperitoneal administration of methyl mercury

A sensitive histochemical technique has been used to visualize the ultrastructural localization of mercury in the anterior pituitary of rats which have been exposed to methyl mercury. After administration of methyl mercury in the drinking water (20 mg X l-1 methyl mercury in distilled water) or intraperitoneally (daily dose 100 ug or 200 ug methyl mercury) intracellular accumulations of mercury were found in the lysosomes and granules of secretory cells (somatotrophs, thyrotrophs and corticotrophs). In non-secretory cells (follicular cell and marginal layer cells) mercury deposits were found in lysosomes. In orally treated rats, the number of mercury deposits increased significantly with time up to day 21. In rats exposed intraperitoneally, a continuous increase was seen in intracellular mercury accumulation. Apart from vacuolation of lysosomes, no structural damage was observed in the cells containing mercury.     

Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material

Autophagy (which includes macro-, micro-, and chaperone-mediated autophagy) is an important biological mechanism for degradation of damaged/obsolete macromolecules and organelles. Ageing non-dividing cells, however, progressively accumulate oxidised proteins, defective organelles and intralysosomal lipofuscin inclusions, suggesting inherent insufficiency of autophagy. To learn more about the role of macroautophagy in the turnover of organelles and lipofuscin formation, we inhibited autophagic sequestration with 3-methyladenine (3 MA) in growth-arrested human fibroblasts, a classical model of cellular ageing. Such treatment resulted in a dramatic accumulation of altered lysosomes, displaying lipofuscin-like autofluorescence, as well as in a moderate increase of mitochondria with lowered membrane potential. The size of the late endosomal compartment appeared not to be significantly altered following 3 MA exposure. The accumulation of lipofuscin-like material was enhanced when 3 MA administration was combined with hyperoxia. The findings suggest that macroautophagy is essential for normal turnover of lysosomes. This notion is supported by reports in the literature of lysosomal membrane proteins inside lysosomes and/or late endosomes, as well as lysosomes with active hydrolases within autophagosomes following vinblastine-induced block of fusion between lysosomes and autophagosomes. The data also suggest that specific components of lysosomes, such as membranes and proteins, may be direct sources of lipofuscin.     

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts.

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.     

The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods

Five hydrolytic enzymes (acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and non-specific esterase) have been studied histochemically in the cells of the digestive gland of Mytilus edulis, Helix aspersa , and certain other lamellibranchs and gastropods. All the enzymes studied have basically similar distributions.
In the digestive cells, the enzymes occur in cytoplasmic granules which are believed to be primary lysosomes; in vacuoles which contain phagocytosed food material; and in vacuoles containing lipofuscin granules, which are the residues of digestive activity.
In the basiphil cells of M. edulis , most of the enzymes are localized in a few cytoplasmic granules; non-specific esterase, however, is found throughout the cytoplasm. In the calcium cells of H. aspersa and the other pulmonate gastropods studied, the enzymes are either in cytoplasmic granules, or distributed diffusely throughout the cytoplasm. Acid phosphatase is also found in the calcium spherules, especially in H. aspersa.
In the excretory cells of H. aspersa and the other pulmonates studied, the enzymes are found in granules in the cytoplasm, and in the lipofuscin granules which lie in the vacuoles of these cells.     

Lipofuscin: mechanisms of age-related accumulation and influence on cell function

The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occurring with a rate inversely related to longevity. Lipofuscin is an intralysosomal, polymeric substance, primarily composed of cross-linked protein residues, formed due to iron-catalyzed oxidative processes. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic cells is inevitable, whereas proliferative cells efficiently dilute it during division. The rate of lipofuscin formation can be experimentally manipulated. In cell culture models, oxidative stress (e.g., exposure to 40% ambient oxygen or low molecular weight iron) promotes lipofuscin accumulation, whereas growth at 8% oxygen and treatment with antioxidants or iron-chelators diminish it. Lipofuscin is a fluorochrome and may sensitize lysosomes to visible light, a process potentially important for the pathogenesis of age-related macular degeneration. Lipofuscin-associated iron sensitizes lysosomes to oxidative stress, jeopardizing lysosomal stability and causing apoptosis due to release of lysosomal contents. Lipofuscin accumulation may also diminish autophagocytotic capacity by acting as a sink for newly produced lysosomal enzymes and, therefore, interfere with recycling of cellular components. Lipofuscin, thus, may be much more directly related to cellular degeneration at old age than was hitherto believed.     

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

Summary Islet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.     

Fluorescence spectra of the SIF cells in rat neuroganglia

The spectral curves of emission of paraform-induced fluorophores in small, intensely fluorescent (SIF) cells in lumbar ganglia of the sympathetic trunk and in the major pelvic ganglion were compared with the fluorescence spectra of lipofuscin granules in the perikaryons of the neurons of the vagus inferior ganglion. As a rule, the fluorescence spectra of SIF cells correlate with the content in them of catecholamines. The spectral characteristics of fluorophores of so-called "yellow" SIF cells have much in common with the fluorescence spectra of lipofuscin granules. Apparently, in some of cases small cells containing lipofuscin may be identified as "yellow" SIF cells.     

Lipofuscin

Over time, postmitotic cells accumulate a non-degradable intralysosomal substance, lipofuscin, which forms due to iron-catalyzed oxidation/polymerization of protein and lipid residues. Lipofuscin is often considered a hallmark of aging, showing an accumulation rate that inversely correlates with longevity. There is an emerging impression that lipofuscin, although still typically considered a harmless wear-and-tear product, may have multiple negative effects. By interfering with the important autophagic process, by which most worn out cellular components are degraded, it may prevent cellular renewal and advance the accumulation of damaged cellular constituents. Due to binding of transition metals, such as iron and copper, lipofuscin also seems to sensitize lysosomes and cells to oxidative stress. Of importance for the pathogenesis of age-related macular degeneration, lipofuscin deposition interferes with the phagocytic activity of retinal pigment epithelial cells and also sensitizes their lysosomes to blue light.