Experimental Gerontology 34 (1999) 943–957

Ceroid/lipofuscin-loaded human ﬁbroblasts show
decreased survival time and diminished
autophagocytosis during amino acid starvation૾
Alexei Termana,b,*, Helge Dalenc, Ulf T. Brunka
a

Division of Pathology II, Department of Neuroscience and Locomotion, Faculty of Health Sciences,
Linkoping University, S-58185 Linkoping, Sweden
¨
¨
b
Institute of Gerontology AMS of Ukraine, Kiev 254114, Ukraine
c
Department of Pathology, The Gade Institute, University of Bergen, Bergen N-5021, Norway
Received 21 June 1999; received in revised form 23 August 1999; accepted 9 September 1999

Abstract
To test whether heavy accumulation of ceroid/lipofuscin can disturb important functions of the
lysosomal system, AG-1518 human ﬁbroblasts, ceroid/lipofuscin-loaded (following prolonged culture at normobaric hyperoxia) or not, were exposed to amino acid starvation. Ceroid/lipofuscinloading resulted in decreased cellular survival. Also, there was an inverse relationship between
amounts of ceroid/lipofuscin and the survival time of individual cells within the same cultures.
Ceroid/lipofuscin-loaded ﬁbroblasts displayed diminished autophagocytotic capacity, as demonstrated by electron microscopy and by treatment of cell cultures with NH4Cl (which inhibits
autophagocytotic degradation by increasing intralysosomal pH) for 1 week before ensuing starvation. The latter treatment increased survival of control cells (due to deposition of nondegraded
autophagocytosed material before start of starvation), but not that of ceroid/lipofuscin-loaded cells.
Moreover, when NH4Cl treatment was combined with starvation, both groups of cells showed
approximately the same shortened survival times, testifying to the causal relationship between
diminished autophagocytosis and decreased survival of starving ceroid/lipofuscin-loaded cells. We
hypothesize that large amounts of undegradable ceroid/lipofuscin within the acidic vacuolar compartment may interfere with lysosomal function, resulting in poor renewal of long-lived proteins and
worn-out/damaged organelles, decreased adaptability, and cell death. © 1999 Elsevier Science Inc.
All rights reserved.
Keywords: Aging; Autophagocytosis; Fibroblasts; Lipofuscin; Lysosomal degradation; Starvation

૾ This study was supported by the Swedish Medical Research Council Grant 4481.
* Corresponding author. Tel.: ϩ46-13-221515; fax: ϩ46-13-221529.
E-mail address: alete@pat.liu.se (A. Terman)
0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 7 0 - 4

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1. Introduction
Lipofuscin is an electron-dense, autoﬂuorescent, polymeric substance that, as a recognized hallmark of aging, accumulates progressively within the lysosomal compartment of
postmitotic cells (Hannover, 1842; Koneff, 1886; Strehler et al., 1959; Sohal and Wolfe,
1986; Harman, 1989; Porta, 1991). Evidence suggests that lipofuscin is formed from
autophagocytosed macromolecular substances by iron-catalyzed oxidative modiﬁcation
(Brunk et al., 1992). Potentially, it can accumulate in any type of cell, not only in
postmitotic ones, and originate from a variety of biomolecules. The amount of lipofuscin
increases in cultured ﬁbroblasts, and other cells when their division is inhibited at
conﬂuency or after repeated passages upon entering phase III (Hayﬂick, 1965; Robbins et
al., 1970; Brock and Hay, 1971; Brandes et al., 1972; Brunk et al., 1973; Rattan et al.,
1982; von Zglinicki et al., 1995). A pigment of almost the same morphological and
physicochemical properties accumulates in vivo in a variety of cell types under pathological conditions, such as malnutrition, stress, ionizing radiation, atherosclerosis, lysosomal storage diseases, etc. (reviewed in Yin, 1996; Terman and Brunk, 1998b). This
latter pigment is usually called ‘lipofuscin of ceroid type’ or simply ‘ceroid’ and basically
has the same origin as lipofuscin.
Although the accumulation of lipofuscin/ceroid is undoubtedly an important manifestation of aging, as well as a number of diseases, it is still unclear whether large amounts
of the pigment can substantially disturb functions of the acidic vacuolar apparatus,
threatening the life of the cell. In elderly individuals, the amount of lipofuscin in some
postmitotic cells may increase dramatically (up to 75% of the cytoplasmic volume) (Treff,
1974). In this case, lipofuscin may occupy a large portion of intralysosomal space, and
probably divert lysosomal enzymatic activity from useful purposes, causing newly produced digestive enzymes to be delivered to lipofuscin-loaded lysosomes rather than to
autophagosomes (Brunk and Terman, 1999). If so, ceroid/lipofuscin-loaded cells would
show enhanced sensitivity to inﬂuences that place increased demands upon the lysosomal
apparatus, e.g., enhanced autophagocytosis.
To test whether this hypothesis is valid, we studied the survival of cultured human
ﬁbroblasts containing different amounts of ceroid/lipofuscin during amino acid starvation.
Under starvation conditions, cells degrade endogenous proteins, thereby generating a pool
of amino acids for critical protein synthesis (Mortimore et al., 1983; Lawrence and Brown,
1992; Blommaart et al., 1997). Some, mainly short-lived, cellular proteins are known to
be degraded in the cytosol (mainly by proteasomes), whereas many other (mainly longlived) proteins and those within organelles undergo intralysosomal degradation (Dice,
1989; Seglen and Bohley, 1992; Berg et al., 1995; Mortimore et al., 1996; Blommaart et
al., 1997). Our investigations were aimed at clarifying the importance of intralysosomal
degradation upon survival of starving cells and the possible effects of lipofuscin accumulation on those functions.

2. Materials and methods
2.1. Culture conditions and experimental design
Human foreskin AG-1518 ﬁbroblasts were obtained at passage 8 from the Coriell
Institute (Camden, NJ, USA) and grown in 25-cm2 plastic ﬂasks (Costar, Cambridge, MA,

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USA) in Eagle’s minimum essential medium in a humidiﬁed atmosphere of 8% O2,
87% N2, and 5% CO2 at 37° by using B 5060 EC/O2 incubators (Heraeus, Hanau,
Germany). The culture medium, supplemented with 10% fetal bovine serum, 2 mM
glutamine, 50 IU/mL penicillin-G, and 50 mg/mL streptomycin, was changed twice a
week. The cells were subcultured by trypsinization at a ratio of 1 : 2. At passage 25,
they were transferred to 35-mm plastic Petri dishes (Costar), and conﬂuent cultures
were established.
For ceroid/lipofuscin loading, the cultures were exposed to normobaric hyperoxia (40%
O2, 55% N2, and 5% CO2) for 4 months, while control cells were maintained under normal
culture conditions (8% O2, 87% N2, and 5% CO2).
For amino acid starvation, trypsinized ﬁbroblast cultures, both control and ceroid/
lipofuscin-loaded (Groups i and ii, respectively), were plated at a density of 5000 cells per
cm2. After 8 h, when the cells were well spread, Eagle’s medium was replaced with Earl’s
buffered salt solution supplemented with 5% fetal calf serum, vitamins, and antibiotics.
The cells did not divide under these conditions. Part of the cultures undergoing starvation
(Group iii, control cells and Group iv, ceroid/lipofuscin-loaded cells) also were exposed
to 10 mM NH4Cl to block intralysosomal degradation. Other cultures (Groups v and vi,
respectively) initially were treated with NH4Cl (under otherwise normal culture conditions) for 1 week, then trypsinized, and ﬁnally exposed to amino acid starvation without
NH4Cl.
Separate dishes from Groups i and ii were used to compare the survival times of
individual cells containing different amounts of ceroid/lipofuscin. This allowed us to
directly relate survival time to the amount of ceroid/lipofuscin and to exclude any possible
effect of the hyperoxia before starvation. Moreover, having sequences of images of the
very same cells we also were able to estimate whether the amount of ceroid/lipofuscin
changed during starvation, i.e., was degraded under enhanced autophagocytosis. The
experiments were performed in triplicate.
2.2. Conventional light microscopy
The cultures were observed by using a Nikon TMS phase-contrast inverted microscope
(Tokyo, Japan). Cells in marked areas of the dishes were counted daily until they died.
Dead cells were recognized by their sharply outlined nuclei, prominent nucleoli, visible
cytoplasmic ﬁlamental structures or by more pronounced signs of damage, such as
destroyed cellular membranes. Vital staining with 0.1% trypan blue for 5 min at room
temperature was used to verify the accuracy of the detection of dead cells in unstained
specimens by phase contrast microscopy (Fig. 1).
Part of the cultures were ﬁxed in 4% neutral buffered formaldehyde with 0.5% Triton
X-100 for 20 min and then stained according to the TUNEL technique for apoptosis (by
using the Apop Tag in situ apoptosis detection kit; Oncor, Gaithersburg, MD, USA) after
the ﬁrst, second, and third days of starvation, respectively. After ﬁxation, the cultures were
subsequently incubated with terminal deoxynucleotidyl transferase and a mixture of
digoxigenin-labeled nucleotides for 1 h and then with anti-digoxigenin-peroxidase-labeled
antibodies for another 30 min. Color development was performed in a 0.05% diaminobenzidine solution with 0.02% H2O2 for 20 min. Nuclei with fragmented DNA stained
brown.

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Fig. 1. Laser scanning images (543-nm excitation light) of human AG-1518 ﬁbroblasts on the third day of amino
acid starvation. (A) Phase-contrast mode; (B) subsequent vital staining with trypan blue, bright ﬁeld mode.
A dead cell with a sharply outlined nucleus, increased granularity, and visible ﬁlamental structures (phase
contrast) was stained with trypan blue, whereas a normal cell remained trypan-blue negative.

2.3. Laser scanning microscopy
To study the survival of individual ﬁbroblasts, images of cells from marked areas of the
dishes were obtained daily by using a LSM-410 inverted laser scanning microscope (Carl
Zeiss, Jena, Germany). A 543 nm (green) helium-neon laser was the source of excitation
light. Two microscope channels were applied: a phase contrast channel for evaluation of
general cell morphology and detection of cell death (by the same criteria as for conventional light microscopy), and a ﬂuorescence channel (590 nm barrier ﬁlter) to visualize
ceroid/lipofuscin. A 63 ϫ 1.25 Plan-Neoﬂuar lens was used for high-resolution confocal
images (pinhole 20). For total cell ceroid/lipofuscin measurements, we used nonconfocal
images (obtained with a 40 ϫ 0.6 LD-Achroplan lens). Pixel values for all images were
within the range of 0 to 255.
2.4. Transmission electron microscopy
For transmission electron microscopy, cells were ﬁxed in situ in a cacodylate-buffered
(0.1 M, pH 7.2) 2% glutaraldehyde solution with 0.1 M sucrose at 4°C, postﬁxed in 1%
osmium tetroxide (in 0.15 M cacodylate buffer) at room temperature, stained en bloc with
2% uranyl acetate in 50% ethanol, dehydrated in a graded series of ethanol, and ﬁnally
embedded in Epon-812. Thin sections were cut with a diamond knife parallel to the

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growth substrate, stained with lead citrate, and examined in a JEOL 1200-EX electron
microscope (Tokyo, Japan). Electron microscopy images were either photographed, or
digitized and stored by using a BioScan wide-angle CCD camera with the attached
software (Gatan, Pleasanton, CA, USA).
2.5. Morphometry
The cell size (area occupied by the cell) and average cellular ceroid/lipofuscin ﬂuorescence intensity were determined on nonconfocal laser scanning images by using the
public domain National Institutes of Health Image program (http://rsb.info.nih.gov/nihimage/). Average ceroid/lipofuscin ﬂuorescence intensity for each cell was measured as
average pixel value minus background. The amount of ceroid/lipofuscin per cell was
calculated as the product of average pixel value by cell area and expressed in arbitrary
units (a.u.).
Digitized electron microscopy images (38 pixels/␮m) were used for measurement of
the cytoplasmic volume fraction of vacuolar/lysosomal structures. For this purpose, the
images were combined with a square grid (available at the National Institutes of Health
Image program) with a spacing of 25 pixels, and the points (intersections of lines)
corresponding to the vacuolar/lysosomal structures, and the rest of the cytoplasm, were
counted.
2.6. Mathematical treatment
The survival time of cells was expressed in days, because a higher degree of accuracy
was not possible. For survival analysis, data from three different dishes for each group
were combined into one sample (a total of 433, 335, 383, 323, 389, and 310 cells in
Groups i, ii, iii, iv, v, and vi, respectively). Combined samples of smaller sizes were used
to study the relationship between the amount of ceroid/lipofuscin and survival time within
Groups i or ii (a total of 72 and 81 cells from three separate dishes, respectively). Because
the distribution of ceroid/lipofuscin between cells was appreciably skewed, its amount was
characterized by medians and interquartile ranges (IQR), rather than by means (averages)
and standard deviations.
Volume fractions of cellular vacuolar/lysosomal structures were determined for each
specimen as a ratio of their point scores to the cytoplasm’s point score (Weibel, 1969), and
then averaged within each group. Fifteen randomly selected cells from each specimen
were analyzed (i.e., a total of 45 cells from each group).
Comparison of survival curves was performed by using the log rank test. In order to
study the relationship between the amount of ceroid/lipofuscin and the survival time
within Groups i and ii, cells were distributed into subgroups according to their survival
times. These subgroups were compared by the Kruskal–Wallis rank test. Kendall rank
correlation coefﬁcients were used to analyze the relationship between median ceroid/
lipofuscin value and survival time of the subgroups. The paired Wilcoxon test was used
to compare ceroid/lipofuscin values of the same cells during starvation. The volume
fractions of the vacuolar/lysosomal structures were compared by using analysis of variance and Student’s t-test.
Values are presented as median (IQR) or as mean Ϯ SD. p Values Ͻ 0.05 were
considered signiﬁcant.

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3. Results
3.1. Amino acid starvation
Starvation of ﬁbroblasts, ceroid/lipofuscin-loaded or not, was associated with shortening and disappearance of their lamellopodiae, some cellular shrinkage, and an increasing
amount of cytoplasmic vesicles. These changes were ﬁrst detectable by phase contrast
microscopy a few hours after the onset of starvation and became more pronounced over
time (Fig. 2). As seen by electron microscopy, ﬁbroblasts starved for 1 day accumulated
numerous autophagic vacuoles of different sizes, which rarely occurred in nonstarving
cells (Figs. 3,A, B, D, and E and 4). These starvation-induced autophagic vacuoles were
recognized by the presence of electron-lucent areas within the vacuoles (apparently due to
increased osmotic pressure with resultant inﬂux of water due to degradation of autophagocytosed structures). Either the whole vacuole was electron-lucent, or it contained both
electron-lucent and electron-dense material, or there was electron-lucent space around
sequestered cytoplasmic structures. It is understood that some autophagic vacuoles may be
early autophagosomes whereas others may be late autophagolysosomes. After 3 days, the
autophagic vacuoles appeared to be more numerous (Fig. 3, C and F and 4). During
starvation, the cells gradually decreased in size and ﬁnally died by necrosis. Although
surface blebbing and TUNEL-positivity (indicative of apoptosis) usually preceded cell
death, dying cells did not show nuclear pyknosis or fragmentation. Most dead cells
remained spread and attached to the bottom of the culture dishes. Ultrastructurally, they
were characterized by swollen nuclei and cytoplasmic alterations typical of necrosis (Fig.
5).
3.2. Ceroid/lipofuscin-loading of cultured ﬁbroblasts shortens their survival during
amino acid starvation
As is evident from Fig. 6, ceroid/lipofuscin-loaded ﬁbroblasts (Group ii), with a median
pigment content of 17.79 (40) a.u., showed signiﬁcantly poorer survival than the control
cells (Group i) having a median ceroid/lipofuscin content of 3.99 (3.31) a.u.
Because the amount of ceroid/lipofuscin in individual cells within the same dishes
varied widely, we also studied its relationship to survival time during starvation. As shown
in Fig. 7A, the shorter survival times of Group ii cells (ceroid/lipofuscin-loaded) were
associated with higher median ceroid/lipofuscin values. This was not the case for Group
i, which had a low intracellular pigment content (Fig. 7B).
3.3. Inhibition of autophagocytotic degradation during starvation equalizes survival
times of ceroid/lipofuscin-loaded and nonloaded ﬁbroblasts
Administration of NH4Cl, which inhibits intralysosomal degradation due to alkalization of the intralysosomal milieu, markedly shortened the survival time of all starved
cultures. No signiﬁcant difference in survival was found between ceroid/lipofuscin-loaded
versus control cells (Groups iv and iii, respectively) when exposed to NH4Cl during
starvation (Fig. 6A).

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Fig. 2. Laser scanning images (543-nm excitation light) of a single ceroid/lipofuscin-loaded ﬁbroblast exposed
to amino acid starvation and followed for 4 days. (A–E) Phase-contrast mode; (F–G) ﬂuorescent mode. Images
were obtained before starvation (A, F) as well as after 1 (B, G), 2 (C, H), 3 (D, I), and 4 (E, J) days of starvation,
respectively. The images E and J show the cell to be dead. Note the increasing amount of cytoplasmic vesicles
(A–D) and the absence of obvious changes in the content of ceroid/lipofuscin (F–I) during starvation.

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Fig. 3. Ultrastructure of control (A–C) and ceroid/lipofuscin-loaded (D–F) ﬁbroblasts: (A, D) under normal
conditions; (B, E) after 1 day; and (C, F) after 3 days of amino acid starvation, respectively. Note that starving
ceroid/lipofuscin-loaded cells accumulate fewer autophagic vacuoles compared to controls. CL, ceroid/lipofuscin-containing lysosomes (of different size and density, and with crystalloid and myelin ﬁgures); AV, autophagic
vacuoles with electron-lucent areas; L, lipid droplets.

3.4. Inhibition of autophagocytotic degradation before starvation selectively prolongs
survival time of ﬁbroblasts with low amounts of ceroid/lipofuscin
As shown in Fig. 6B, exposure to NH4Cl, under otherwise normal culture conditions,
for 1 week before the start of starvation markedly prolonged the survival time of
ﬁbroblasts with a low pigment content (Group v). In contrast, pretreatment of ceroid/
lipofuscin-loaded ﬁbroblasts with NH4Cl (Group vi) did not result in any increase of the
survival time.
3.5. Starvation-induced autophagocytosis is depressed in ceroid/lipofuscin-loaded
ﬁbroblasts
Ultrastructurally, human ﬁbroblasts exposed to prolonged normobaric hyperoxia
(Group ii) accumulated numerous irregularly shaped lysosomes of variable size and
density, often with crystalloid or myelin-like structures. Apparently, most of them con-

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Fig. 4. Volume fraction of autophagic vacuoles (electron-lucent areas) in ﬁbroblasts cultivated under normal
conditions and exposed to amino acid starvation for 1 and 3 days. White columns, control cells (Group i); dark
columns, ceroid/lipofuscin-loaded cells (Group ii). Although the amount of autophagic vacuoles was initially
higher in ceroid/lipofuscin-loaded cells, its increase due to starvation was much less than in controls. Volume
fractions of autophagic vacuoles signiﬁcantly differ from each other within Groups i or ii (analysis of variance,
p Ͻ 0.0001 for both groups), as well as between Groups i and ii (t-test; p ϭ 0.0036, p ϭ 0.0021, and p Ͻ 0.001
for cells under normal conditions, and after 1 and 3 days of starvation, respectively).

tained autoﬂuorescent material and could be classiﬁed as ceroid/lipofuscin inclusions.
Control cells, not exposed to hyperoxia (Group i) accumulated much less pigment (see
also Nilsson and Yin, 1997; Terman and Brunk, 1998a). One day after the onset of
starvation, the number and volume fraction of newly formed autophagic vacuoles (i.e.,
with electron-lucent contents) increased, but to a much lesser degree than in control cells
(Figs. 3, B and E and 4). After 3 days, the number and size of these structures had
increased further, although less dramatically than in the cells of Group i (Figs. 3, C and
F and 4). It is remarkable that under normal conditions the amount of autophagic vacuoles
in ceroid/lipofuscin-loaded cells was higher than in controls (Fig. 4). This might reﬂect a
prolonged degradation time of autophagic structures in ceroid/lipofuscin-loaded cells.
3.6. Starving cells fail to degrade ceroid/lipofuscin inclusions
Although ceroid/lipofuscin was frequently seen within newly formed autophagic vacuoles (having electron-lucent spaces) of starving ﬁbroblasts, it was preserved with respect
to its characteristic features (medium to high electron density, presence of crystalloid or
myelin ﬁgures) under starvation. In cells starved for 3 days, the ceroid/lipofuscincontaining lysosomes tended to aggregate and fuse, however without any substantial
change of the pigment ultrastructure.
Ceroid/lipofuscin autoﬂuorescence did not decrease during amino acid starvation, as
was evident from sequential images of the same cell (Fig. 2), as well as from measurement
of cellular autoﬂuorescence before starvation and on the third day of cultivation without

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Fig. 5. Electron micrograph of a dead cell after 3 days of starvation. Note nuclear and cytoplasmic deorganization
and swelling, ﬁndings indicative of necrosis. N, nucleus; L, lipid droplets.

amino acids [median values of cellular autoﬂuorescence were 14.85 (46.38) a.u. versus
16.55 (47.78) a.u., respectively; n ϭ 30; p ϭ 0.869].

4. Discussion
Although amino acid starvation may not be physiologically relevant, this starvation
model, nevertheless, allows us to detect any negative inﬂuences of ceroid/lipofuscin on
lysosomal functions.
As is evident from the results, ﬁbroblasts from ceroid/lipofuscin-loaded cultures
(Group ii) have a shortened survival time during amino acid starvation when compared to
cells from control cultures (Group i). Because ceroid/lipofuscin loading was a result of
prolonged oxidative stress, an effect of hyperoxia on cell survival under ensuing starvation
cannot be ruled out. However, a comparison of cells with different amounts of pigment
within Group ii (i.e., where all the cells were under identical conditions) yielded the same
results. That is, cells bearing the least amounts of ceroid-lipofuscin had a clear survival
advantage. The observed negative correlation between the content of ceroid/lipofuscin and
cell survival time (as any other association between variables) does not necessarily prove
a causal relationship. However, because cell survival during starvation is known to depend
on lysosomal degradation activity (Mortimore et al., 1983; Lawrence and Brown, 1992;
Blommaart et al., 1997), there are, indeed, good reasons to believe that cells whose
lysosomes are occupied by undegradable ceroid/lipofuscin granules, cannot efﬁciently
autophagocytose, and, therefore, show decreased survival time.
Additionally, this belief is supported by our NH4Cl experiments. This compound is
known to inhibit intralysosomal, but not proteasome degradation (Kopitz et al., 1993; El
Khissiin and Leclercq, 1999). Both ceroid/lipofuscin-loaded and control cells treated with
NH4Cl during amino acid starvation (Groups iv and iii, respectively) showed approxi-

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Fig. 6. (A) Survival curves of control and ceroid/lipofuscin-loaded ﬁbroblasts exposed to amino acid starvation
without (Groups i and ii, respectively) or with (Groups iii and iv, respectively) 10 mM NH4Cl (present during
the whole period of starvation). Statistical signiﬁcance (log rank test): Group i versus Group ii, Group i versus
Group iii, and Group ii versus Group iv, p Ͻ 0.0001; Group iii versus Group iv, p ϭ 0.494. Mean Ϯ SD survival
time in days: Group i, 4.12 Ϯ 1.83; Group ii, 3.18 Ϯ 1.3; Group iii, 1.02 Ϯ 0.74; Group iv, 0.98 Ϯ 0.79. Sample
sizes: 433, 335, 383, and 323 cells in Groups i, ii, iii, and iv, respectively. (B) Survival curves of control and
ceroid/lipofuscin-loaded ﬁbroblasts exposed to amino acid starvation with (Groups v and vi, respectively) or
without (Groups i and ii, respectively) an initial exposure (before start of the starvation) to 10 mM NH4Cl for
1 week. Statistical signiﬁcance (log rank test): Group i versus Group ii and Group i versus Group v, p Ͻ 0.0001;
Group ii versus Group vi, p ϭ 0.53. Mean Ϯ SD survival time in days: Group i, 4.12 Ϯ 1.83; Group ii, 3.18 Ϯ
1.3; Group v, 5.67 Ϯ 2.31; Group vi, 3.05 Ϯ 1.45. Sample sizes: 433, 335, 389, and 310 cells in Groups i, ii,
v, and vi, respectively.

mately the same (foreshortened) survival time. The average survival time decreased 3-fold
for Group iv and 4-fold for Group iii cells (Fig. 6), conﬁrming the importance of
intralysosomal degradation as a source of amino acids for the survival of starving cells.
Because suppression of lysosome-dependent protein degradation equalized survival times
for Groups iii and iv, it is reasonable to suppose that the difference in survival between
starving ceroid/lipofuscin-loaded and control cells was principally determined by differences in lysosomal degradative capacity, and that the proteasome pathway of proteolysis
is of much less importance than the lysosomal one in enabling the recycling of endogenous protein-derived amino acids during starvation.
In contrast, inhibition of intralysosomal degradation with NH4Cl before starvation
prolonged the survival time of control cells (Group v), most likely due to an accumulation
of a pool of nutrients within the lysosomes which later could be used during the ensuing
starvation. The fact that NH4Cl pretreatment did not prolong the survival time of ceroid/
lipofuscin-loaded ﬁbroblasts (Group vi) indirectly testiﬁes to the limited autophagocytotic
capacity of these cells.
As shown by electron microscopy, starvation of ﬁbroblasts is associated with the
appearance of numerous autophagic vacuoles. Apparently partial degradation of the cells’
own components (mainly proteins) provides amino acids for essential protein synthesis.
Autophagic vacuoles were markedly less numerous in starved ceroid/lipofuscin-loaded
cells than in control cells. Obviously, ceroid/lipofuscin-rich cells cannot efﬁciently degrade their own components in order to sustain life during starvation. It should, however,

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Fig. 7. (A) “Box and whiskers” plot graph showing ceroid/lipofuscin content of ﬁbroblasts of Group ii
(pigment-loaded) that survived for different times during amino acid starvation. Bold horizontal bars, tops and
bottoms of the boxes, and whiskers show the medians, upper and lower quartiles, and upper and lower deciles,
respectively. n, number of cells in each interval. Note that a longer survival time corresponds to a lower median
content of ceroid/lipofuscin. Kruskall–Wallis rank test: p ϭ 0.022. Kendall rank correlation: tau ϭ Ϫ1, p ϭ
0.042. (B) “Box and whiskers” plot graph showing ceroid/lipofuscin content of ﬁbroblasts of Group i
(control cells) that survived for different times during amino acid starvation. No signiﬁcant relationship
between survival time and the ceroid/lipofuscin amount (always low compared to the pigment-loaded
group) was found. Kruskall–Wallis rank test: p ϭ 0.881. Kendall rank correlation: tau ϭ Ϫ0.2, p ϭ 0.624
(compare with A).

be stressed that several ceroid/lipofuscin-containing lysosomes of starved cells showed
signs of some autophagic activity in the form of electron-lucent, membrane-limited
spaces. This points out that ceroid/lipofuscin-containing lysosomes are integrated parts of
the lysosomal apparatus with a capacity for fusion and ﬁssion reactions involving autophagosomes (Brunk, 1973).
That ceroid/lipofuscin is generally undegradable was recently demonstrated in several
studies (Elleder et al., 1995; Terman and Brunk, 1998c; Terman and Brunk, 1998a). Here
we have shown that also under conditions of cellular starvation, when autophagocytosis
is highly activated, cells are still unable to degrade ceroid/lipofuscin. As we found in an
earlier study (Terman et al., 1999), ceroid/lipofuscin-rich ﬁbroblasts have an expanded
lysosomal compartment with increased amounts of cathepsin D and most other lysosomal
enzymes. We consider this increased production of lysosomal enzymes to be a consequence of the cells’ futile attempts to degrade the undegradable pigment. For statistical
reasons, most newly produced lysosomal enzymes—transported from the Golgi area in
small vesicles—would end up in ceroid/lipofuscin-containing lysosomes rather than in
newly formed autophagosomes. Because the production of lysosomal enzymes is limited,
cells with high quantities of ceroid/lipofuscin would not be able to sufﬁciently increase
autophagocytotic activity when necessary.
Starvation is one example of a situation when demands on the lysosomal system are

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Fig. 8. The arrows symbolize the ﬂow of lysosomal enzymes from Golgi apparatus to autophagolysosomes (A),
or to lipofuscin-loaded lysosomes (L). T stands for the total production of lysosomal enzymes. Mitochondria and
other cellular structures are normally constantly renewed thanks to autophagocytotic activity. In aged postmitotic
cells the lysosomal compartment is expanded, and to a large extent occupied, by undegradable lipofuscin that is
considered to cause a compensatory increased production of lysosomal enzymes (most of which are wasted by
being directed to the lipofuscin-loaded lysosomes) in order to provide enough capacity for necessary autophagocytosis (increased T, allowing A to remain of normal size even when L increased). Because the production
of lysosomal enzymes is limited, extremely lipofuscin-loaded senescent cells will be unable to provide their
autophagocytotic machinery with enough lysosomal enzymes for proper degradation (T is now at its maximum
resulting in diminishing of A when L is increased). Consequently, such old cells would contain abundant
worn-out and damaged mitochondria, which are known to produce increased amounts of reactive oxygen
species. As a result, senescent cells will die due to energy deﬁciency and/or oxidative stress.

increased. Other examples are various kinds of cell damage (caused by oxidative stress,
hypoxia, toxic effects, etc.) that require reparative autophagocytosis (Brunk et al., 1995)
and, thus, there are good reasons to expect reduced adaptability of ceroid/lipofuscinloaded cells also under such conditions. Because lipofuscin progressively accumulates
within aging postmitotic cells, their autophagocytotic capacity may ﬁnally decrease to a
level which is not sufﬁcient for cell renewal even under normal conditions. The renewal
of mitochondria is of particular importance, because oxidative damage to these organelles
was shown to be one of the major contributors to aging (Ames et al., 1995). A theoretical
sequence of events, in which ceroid/lipofuscin accumulation leads to cell death by causing
a progressive cellular inability to catabolize damaged mitochondria by autophagocytosis,
is given in Fig. 8. The predicted result would be abnormal survival of damaged mitochondria and increasing oxidative stress because old and damaged mitochondria progressively leak electrons resulting in formation of superoxide and hydrogen peroxide. This
hypothesis, called the “mitochondrial-lysosomal axis theory of cellular aging,” is described in some detail in a separate paper (Brunk and Terman, 1999).
Thus, there are good reasons to believe that ceroid/lipofuscin is not only an undegradable intralysosomal waste substance, but also is potentially harmful to the normal function
of the lysosomal apparatus and increases the probability of death when it accumulates in
large quantities.

956

A. Terman et al. / Experimental Gerontology 34 (1999) 943–957

Acknowledgments
H.D. was the recipient of a visiting scientiﬁc scholarship from the Linkoping Univer¨
sity Hospital. The authors thank Anne Marie Sandsbakk Austarheim, Turid F. Gulbrandsen, and Karin Roberg for technical assistance.
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