Fasting in
1-antitrypsin deficient
liver: constitutive activation of autophagy
Jeffrey H.
Teckman,
Jae-Koo
An,
Scott
Loethen, and
David H.
Perlmutter
Departments of Pediatrics and Cell Biology and Physiology,
St. Louis Children's Hospital, Washington University School of
Medicine, St. Louis, Missouri 63110
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ABSTRACT |
1-Antitrypsin
(
1-AT) deficiency causes severe liver injury in a
subgroup of patients. Liver injury is thought to be caused by retention
of a polymerized mutant
1-ATZ molecule in the
endoplasmic reticulum (ER) of hepatocytes and is associated with an
intense autophagic response. However, there is limited information
about what physiologic stressors might influence liver injury. In this study, we examined the effect of fasting in the PiZ mouse model of
1-AT deficiency, because fasting is a well-characterized
physiological stressor and a known stimulus for autophagy. Results show
that there is a marked increase in fat accumulation and in
1-AT-containing globules in the liver of the PiZ mouse
induced by fasting. Although fasting induced a marked autophagic
response in wild-type mice, the autophagic response was already
activated in PiZ mice and did not further increase with fasting. PiZ
mice also had a significantly decreased tolerance for prolonged
fasting compared with wild-type mice (PiZ mice 0% survival of 72-h
fast; wild-type 100% survivial). These results demonstrate an altered
response to stress in the
1-AT-deficient liver,
including inability to further increase an activated autophagic
response, a developmental state-specific increase in
1-AT-containing globules, and increased mortality.
protein degradation; endoplasmic reticulum
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INTRODUCTION |
THE CLASSICAL FORM of
1-antitrypsin (
1-AT) deficiency,
homozygous PIZZ
1-AT deficiency, is caused by a point
mutation encoding a substitution of lysine for glutamate-342
(23). This substitution confers polymerogenic
properties on the mutant
1-ATZ molecule (2). Aggregated mutant
1-ATZ is retained in
the endoplasmic reticulum (ER) rather than secreted in the blood and
body fluids where its function is to inhibit neutrophil proteases.
Individuals with this deficiency have a markedly increased risk of
developing emphysema by a loss-of-function mechanism, i.e., reduced
levels of
1-AT in the lung to inhibit connective tissue
breakdown by neutrophil elastase, cathepsin G, and proteinase 3. A
subgroup of PIZZ individuals develops liver injury and hepatocellular
carcinoma by a gain-of-function mechanism, i.e., accumulation of
aggregated mutant
1-ATZ within the ER is toxic to liver
cells. The "accumulation" mechanism is best demonstrated by
transgenic mice engineered for expression of the human
1-ATZ gene (1, 4). In addition to periodic
acid-Schiff (PAS)-positive, diastase-resistant intrahepatic globules
that represent ER dilated with the aggregated mutant protein, these
mice develop liver injury and hepatocellular carcinoma. Because there
are normal levels of antiproteases in these animals, as directed by
endogenous genes, the liver injury cannot be attributed to a
loss-of-function mechanism. In fact, detailed histological characterization of the liver in one transgenic mouse model by Geller
and colleagues (5) has shown that there are focal areas of
inflammatory infiltration and regenerative activity in the form of
multicellular liver plates (5). As these mice reach 18 mo
of age, close to 80% of them have adenomas and carcinomas in the liver.
There is marked variability in the phenotypic expression of liver
disease among affected PIZZ individuals. Some patients have severe
liver disease and need liver transplantation surgery early in life,
whereas others do not develop clinical signs of liver disease until
late in adult life, if ever. A prospective nationwide screening study
in Sweden has shown 80-90% of PIZZ individuals have no clinical
evidence of liver disease at 18 years of age, their age at the time of
the last report on the population (20, 21). One study
(26) with genetically engineered fibroblast cell lines
from PIZZ individuals carefully characterized for the absence or
presence of liver disease indicates that protection from liver disease
is correlated with efficient degradation of
1-ATZ in the
ER and, therein, presumably with a reduced burden of aggregated protein
in the ER. However, there is relatively limited information in the
literature about the genetic and environmental mechanisms for
inefficient degradation or for the effect of physiological stressors on
susceptibility to liver injury. There is also very little information
about the mechanisms responsible for the effect of developmental stage
on liver injury in
1-AT-deficient patients, particularly
the observation of exacerbated hepatic inflammation and dysfunction in
the newborn followed by a honeymoon period until late childhood/early
adolescence in many patients.
Recent studies in our laboratories have demonstrated that ER retention
of mutant
1-ATZ induces a marked autophagic response in
cell culture and PiZ transgenic mouse models of
1-AT
deficiency as well as in the liver of PIZZ patients (24).
The autophagic response is thought to be a general mechanism whereby
cytosol and intracellular organelles, such as ER, are first sequestered from the rest of the cytoplasm within unique vacuoles and then degraded
by fusion with lysosomes to clear the cell of senescent constituents.
It occurs in many cell types during the cellular remodeling that
accompanies differentiation, morphogenesis, and aging and is induced by
stress states such as nutrient deprivation. In this study, we examined
the effect of fasting on the liver of the PiZ mouse, because it is a
well-defined physiological stimulus of autophagy and a known
environmental stressor of liver disease in infants and children.
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MATERIALS AND METHODS |
Materials.
Antibodies against
1-AT included rabbit anti-human
1-AT from DAKO (Santa Barbara, CA) and goat anti-human
1-AT from Cappel (Durham, NC). Antibodies against
calnexin SPA-865 and antibodies against BiP/GRP78 were purchased from
StressGen (Victoria, BC). Antibodies against lysosome-associated
membrane protein-1 (LAMP-1) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), and antibodies against cathepsin were
from Upstate Biotechnology (Lake Placid, NY). Rhodamine and Cy2
conjugated anti-Ig antibodies were purchased from Jackson
Immunoresearch (West Grove, PA).
Mice.
Liver tissue was removed by conventional surgical techniques from
PiZ and C57 black mice that had been killed with anesthesia. Standardized sections were obtained from the same region of the left
median lobe and fixed for light microscopic, immunofluorescent, or
electron microscopic analysis as previously described
(24). During fasting experiments, mice were kept in their
usual cages with water available ad libitum but without any source of
solid food or nutrients. An 18-h interval of fasting to study autophagy was selected on the basis of previous studies of autophagy in rodent
liver (3, 8, 9, 17, 18). All studies were approved by the
Washington University Animal Studies Committee.
Microscopy.
At the time of death, liver sections were fixed in formalin, paraffin
embedded, and sectioned for light and immunofluorescent microscopy
using previously described techniques (24).
Well-established techniques were used for immunostaining and
hematoxylin and eosin (H & E), PAS/digestion, and oil red-O staining.
All PAS studies were performed in parallel with nondigested controls.
All double-label immunostains were repeated using different secondary
antibodies with different fluorophores to confirm colocalization
results. Samples for transmission electron microscopy were fixed at the time of death in 1% glutaraldehyde-0.1 M sodium cacodylate and embedded in polybed for ultrathin section transmission electron microscopy, as described previously (24). All light and
fluorescent photomicrographs were obtained by viewing with a Zeiss
Axioskope microscope with the Axiocam digital image capture system.
Electron microscopy specimens were viewed and photographed using a
Zeiss 902 transmission electron microscope. Quantification of
PAS-positive globules in liver was performed by averaging the total
globules counted in five randomly selected fields with a ×20 objective lens for each specimen. Quantification of autophagy was performed with
grids superimposed on 10 photomicrographs of each specimen at ×3,000
and showing a complete hepatocyte with a nucleus. The area of cytoplasm
occupied by autophagic vacuoles was then determined as a percentage of
the total area of cytoplasm (not including the area occupied by fat
droplets). Immune label electron microscopy was performed exactly as
previously described (24). All studies involving human
tissue were approved by the Washington University Human Studies Committee.
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RESULTS |
Histological features of PiZ transgenic mouse liver at baseline and
after fasting.
First, we examined sections of liver from 3-mo-old PiZ mice at baseline
and after an 18-h fast (Fig. 1).
Representative photomicrographs of liver stained with H & E are shown
in Fig. 1A, left, and demonstrate the focal
lymphocytic infiltration of hepatic lobules, as previously described in
these mice (1). The hepatic architecture is otherwise preserved, and the hepatocytes appear intact except for round, eosinophillic bodies identifiable within the cytoplasm of some hepatocytes. These bodies were more fully examined with the PAS stain
described below. Examination of the fasted liver specimens by H & E
stain revealed no significant differences in the inflammatory infiltrate or overall hepatic architecture, although diffuse
vacuolization was observed within the cytoplasm of all hepatocytes,
suggesting the possibility of microvesicular fat deposition.
Eosinophillic bodies within the cytoplasm of some hepatocytes were
observed again.

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Fig. 1.
Light microscopic analysis of PiZ and C57 black liver at baseline
and after fasting. Livers from 3-mo-old PiZ and C57 black mice at
baseline and after an 18-h fast were fixed and paraffin-embedded using
standard techniques and then sectioned for staining by hematoxylin and
eosin (H & E) and periodic acid-Schiff (PAS)/digestion (arrows point to
inflammatory infiltrates; A) and oil red-O (B) as
shown (arrow points to fat droplet). Bar = 10 µm.
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Next, we examined these same specimens with PAS staining followed by
digestion (Fig. 1A, middle). This technique
stains glycoprotein red and has been previously shown to label
accumulation of mutant
1-ATZ within dilated ER membranes
in hepatocytes (6, 12, 15, 19, 24, 28, 11). These
structures have been termed "globules" when described in
1-AT-deficient liver and in the PiZ mouse liver. The
presence of mutant
1-ATZ in these globules has been
confirmed by immunostaining in this (see Immunofluorescence analysis for
1-AT in the PiZ mouse liver before and
after fasting) and in many previous studies (6, 11, 12, 15,
19, 28). The globules also immunostain positive for known ER
resident proteins and immunostain negative for lysosomal markers,
consistent with their identification as ER membranes and not lysosomes
or autophagosomes (see Immunofluorescence analysis for
1-AT in the PiZ mouse liver before and after
fasting). The results here show obvious PAS-positive globules within the cytoplasm of many but not all hepatocytes. However,
examination of the liver after fasting revealed a marked increase in
the number of PAS-positive globules. Quantitation of the number of
globules per microscopic field in the liver of five 3-mo-old PiZ mice
after 18 h of fasting revealed a 2.5-fold increase over the number
observed at baseline (P < 0.05 by ANOVA). Control, C57
black mouse livers stained with PAS/digestion showed no globular
accumulations of glycoprotein either at baseline or after fasting (Fig.
1A, right).
Because the H & E-stained liver sections suggested the presence of
increased microvesicular fat, we also examined these same liver
specimens with oil red-O stain to label fat accumulation within
hepatocytes (Fig. 1B). Nontransgenic mice of the same
genetic background (C57 black) were used as control. The results show that there is an increase in fat deposition in C57 black mice after
fasting for 18 h. For the PiZ mouse, there is a similar amount of
fat at baseline but a much more dramatic increase after fasting. Taken
together, these results indicate that there is a specific alteration in
the response of the PiZ mouse liver to fasting characterized by
increased PAS-positive, diastase-resistant globules, and increased steatosis.
Effect of developmental stage on the response of the PiZ
mouse liver to fasting.
Next, we examined PiZ mouse liver specimens for changes in
histology and
1-ATZ accumulation associated with aging.
Liver specimens from at least three individual mice at ages of 1, 2, 3, 4, 6, 9, 12, and 16 mo were examined by PAS stain with digestion with
representative specimens from 1-, 4-, 9-, and 16-mo-old mice shown in
Fig. 2. The results show a progressive
increase in the size of the globules during aging. Mean globule
diameter was 1.7 ± 0.8 (SD) µm at 1 mo of age vs. 6.3 ± 3 µm at 16 mo of age (P < 0.001). There was no change
in the number of globules, as determined by quantitative analysis.
There was no change in the amount of fat deposition during aging.

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Fig. 2.
Effect of age on PiZ liver. Livers from PiZ mice aged 1, 4, 9, and
16 mo were fixed, paraffin embedded, sectioned, and stained with
PAS/digestion. Bar = 10 µm.
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Next, we examined the effect of fasting at different ages (Fig.
3). The results show the marked 2.5-fold
increase in the number of globules in 3-mo-old mice as described above
but no change in 1- or 10-mo-old mice. Analysis of three individual
mice at ages 1, 2, 3, 4, 6, 9, 10, and 12 mo showed that the increase in number of globules induced by fasting was only present in mice between 2 and 6 mo and peaked at 3 mo of age (data not shown). In
contrast, aging had no effect on the fat deposition induced by fasting
(data not shown). These results indicate that there is at least one
developmental stage-specific alteration in the PiZ mouse liver at
baseline, characterized by a progressive increase in the size of
globules, and a developmental stage-specific alteration in the response
to fasting, characterized by an increased number of globules between 2 and 6 mo of age.

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Fig. 3.
Effect of age and fasting on PAS-positive globules in PiZ liver.
Livers from PiZ mice aged 1, 3, and 10 mo, at baseline, and after an
18-h fast were fixed, sectioned, and stained with PAS/digestion as
shown. Bar = 10 µm.
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We also examined 3-mo-old PiZ and C57 black mice during prolonged
fasting. Groups of five mice in each category were monitored during a
72-h fast. The results showed that none of the PiZ mice (0%) were able
to survive the 72-h period but that all (100%) of the C57 black mice
survived without difficulty. The cause of death was not apparent. None
of the mice that died during the prolonged fast had massive hepatic
necrosis by routine histological examination of the livers. However,
the livers were completely devoid of fat at death (data not shown),
despite the marked fat accumulation noted in the PiZ mice after the
overnight fasting intervals (Fig. 1.)
Immunofluorescence analysis for
1-AT in the PiZ
mouse liver before and after fasting.
Next we used immunofluorescence for
1-AT to analyze the
increase in globules in fasted 3-mo-old PiZ mice (Fig.
4a). The results show, at
baseline, that antibody to
1-AT stains globules
intensely in a manner identical to PAS staining. Moreover, antibody to
1-AT diffusely stains the cytoplasm of many, but not
all, hepatocytes, in a fine reticular pattern. Positive staining with
antibody to
1-AT of some, but not all, hepatocytes is a
characteristic of human PIZZ and mouse PiZ liver (6, 11, 12, 15,
19, 28). After fasting for 18 h, there is an increase in
1-AT-positive globules, exactly as shown with PAS
staining above. Interestingly, fasting was also associated with a
complete disappearance of the diffuse, reticular staining in the
cytoplasm.

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Fig. 4.
A: immunofluorescent staining for
1-antitrypsin (AT) and BiP in PiZ and C57 black liver at
baseline and after an 18-h fast. Livers from 3-mo-old PiZ and C57 black
mice were fixed, sectioned, and immunostained with either primary
antibody to 1-AT or BiP and secondary anti-Ig antibody
conjugated to rhodamine (red) or Cy2 (green). B:
high-magnification views of the same specimens double immunostained
with antibody to 1-AT (green) and cathepsin (Cat) D
(red). White arrows, typical globules; white arrowheads, typical areas
of cytoplasm with the diffuse, fine reticular staining pattern; black
arrows, small, punctate structures positive for cathepsin D; black
arrowheads, small clusters of structures positive for both
1-AT and cathepsin D (yellow). Bar = 10 µm.
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Next, we examined the same specimens for staining with antibody to BiP,
a well-characterized resident protein of the ER (Fig. 4A,
middle). The results show, at baseline, staining of globules and diffuse staining of the cytoplasm of all hepatocytes in a fine,
reticular pattern. After fasting, anti-BiP antibody exclusively stains
globules. The diffuse reticular cytoplasmic staining completely disappears. This change in distribution of
1-AT and BiP
staining induced by fasting was specific for the PiZ mouse, as shown by immunostaining for BiP in nontransgenic C57 black mice (Fig.
4A, bottom). Although there was a slight decrease
in intensity after fasting, the pattern of diffuse, reticular staining
by anti-BiP antibody was the same at baseline and after fasting in the
C57 black mouse. These data indicate that there is a specific increase in globules that stain positively for
1-AT and BiP and a
specific change in distribution of
1-AT and BiP staining
in the PiZ mouse induced by fasting.
To be assured that the globule structures were, in fact, dilated ER and
not themselves autophagosomes, we first performed double-label
immunofluorescence on these same baseline and fasted PiZ liver
specimens for
1-AT and cathepsin D, which is found in
lysosomes and autophagosomes (Fig. 4B, top). The
results show that the globules stain positively for
1-AT
but not for cathepsin D. However, many small, punctate structures
consistent with lysosomes scattered throughout the cytoplasm are
labeled positively for cathepsin D and not
1-AT.
Occasional clusters of very small, punctate structures are double
labeled for
1-AT and cathepsin D, which are consistent
with previously published labeling patterns of nests of autophagosomes
under immunofluorescence and immune electron microscopy (3, 18,
24). Identical staining of control C57 black baseline and fasted
liver specimens (Fig. 4B, bottom) demonstrated
punctate structures positive for cathepsin D but, as expected, no
structures immunoreactive for human
1-AT.
Next, to determine if antibodies to
1-AT and BiP are
staining the same structures, double-label immunostaining was applied to the same specimens (Fig.
5A). The results show that, at
baseline,
1-AT and BiP are colocalized in globules, but
there are some hepatocytes with diffuse reticular staining of the
cytoplasm with anti-BiP but not anti-
1-AT, as previously
described above (Fig. 5A). After fasting,
1-AT and BiP staining is shifted to and colocalized within the globules.

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Fig. 5.
Immunofluorescent double staining of 1-AT and either
BiP (A) or calnexin (B) in PiZ liver at baseline
and after an 18-h fast. Livers from 3-mo-old PiZ mice at baseline and
after fasting were fixed, sectioned, and immunostained with primary
antibody to 1-AT and either BiP or calnexin and then
secondary antibody conjugated to rhodamine for 1-AT
(red) and Cy2 for BiP and calnexin (green). Arrows show
1-AT-negative hepatocytes that are BiP or calnexin
positive. Bar = 10 µm.
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We also used antibody to calnexin, another resident protein of the ER,
on these liver specimens (Fig. 5B). The results were identical. At baseline,
1-AT and calnexin are
colocalized in globules, but there are some hepatocytes with diffuse
reticular staining of the cytoplasm with anti-calnexin but not
anti-
1-AT (Fig. 5B). After fasting,
1-AT and calnexin are exclusively colocalized within the
increased numbers of globules.
The change in distribution of
1-AT, BiP, and
calnexin immunoreactivity was seen in PiZ mice at all ages from 1 to 16 mo (data not shown).
Ultrastructural analysis of the PiZ mouse liver before and after
fasting.
To determine if the disappearance of the diffuse reticular cytoplasmic
immunostaining with anti-
1-AT, anti-BiP, and
anti-calnexin antibodies in PiZ mouse liver after fasting is because of
a disappearance of nondilated ER, we examined the liver of the fasted
PiZ mouse by electron microscopy (Fig.
6). The results show that there are abundant nondilated ER cisternae throughout the cytoplasm in addition to massively dilated globules and many fat droplets. These results indicate that the disappearance of the diffuse reticular cytoplasmic immunostaining for
1-AT, BiP, and calnexin is explained
by a change in the distribution of these molecules rather than a
complete transformation of ER membranes.

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Fig. 6.
Electron microscopy of hepatocytes from 3-mo-old PiZ
liver after an 18-h fast. Liver from a 3-mo-old PiZ mouse after fasting
was fixed, plastic-embedded, and subjected to ultrathin sectioning for
transmission electron microscopy as described. This specimen was
derived from the same PiZ mouse that was used for the specimens shown
in Figs. 3-5. rER, nondilated rough endoplasmic reticulum (ER);
Gl, globule (dilated ER); F, fat droplet; M, mitochondria. Bar = 1 µm. AP, autophagic vacuole
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Finally, we examined the ultrastructure of hepatocytes in PiZ mice
using transmission electron microscopy to determine the effect of
fasting on autophagy. We examined the same baseline and fasted C57
black and PiZ liver specimens as described above by transmission
electron microscopy. Representative photomicrographs are shown
in Fig. 7. At
baseline, the nontransgenic C57 black mouse hepatocytes show normal
structures, including a nucleus, mitochondria, and ER membranes. Some
of these hepatocytes have one or two double or multilammelar vacuoles
containing electron-dense material, which are the classical
morphological characteristics of autophagosomes (Fig. 7, A
and B). When the C57 black mouse is fasted 18 h, there
is a considerable increase in the autophagic vacuoles visible in the
cytoplasm, consistent with previous studies that have demonstrated
increased autophagy during fasting (3, 8, 9, 17, 18).
Fasting is also associated with an increase in scattered, small, simple
cytoplasmic vacuoles containing material that has the density of lipid,
consistent with mild-to-moderate microvesicular fat accumulation
described above in the C57 black mouse. There is little if any change
in the architecture of the ER detectable after fasting.

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Fig. 7.
A and B: transmission electron
microscopy of hepatocytes from PiZ and C57 black liver at baseline and
after fasting. Livers from 3-mo-old PiZ and C57 black mice at baseline
and after an 18-h fast were fixed, plastic embedded, and subjected to
ultra-thin sectioning for electron microscopy as described in
MATERIALS AND METHODS. A: low magnification;
B: high magnification. Arrow, autophagic vacuole; N,
nucleus. Bar = 1 µm. C: immune-label transmission
electron microscopy with antibody against either
lysosomeassociated membrane protein-1 (LAMP-1) or cathepsin
D and secondary anti-Ig antibody bearing immunogold beads. Arrows,
multilamellar membranes; arrowheads, immunogold beads. Bar = 200 nm.
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In the PiZ mouse liver, there are already a large number of
autophagosomes at baseline, which can be identified by the identical ultrastructural criteria as autophagosomes in the C57 mice. In fact,
the electron photomicrographs shown in Fig. 7A suggest that there are significantly more autophagosomes in the PiZ mouse liver at
baseline than in the C57 black mouse liver after fasting.
Interestingly, there is no increase in the number of autophagosomes
when comparing PiZ mouse liver after fasting with that at baseline. The
increase in globules and fat vesicles is readily apparent and
intervenes between autophagosomes, allowing fewer to be visible in any
single field of view. At higher magnification in Fig. 7B,
the morphology of the autophagosomes is even more clearly evident.
Again, there is an increase in the number of autophagosomes after
fasting in the C57 black mouse. There are many more autophagosomes in
the PiZ mouse at baseline without any increase after fasting. To
provide further assurance that these multilamellar vacuoles containing electron-dense debris were indeed autophagosomes, we performed immune-label electron microscopy on the PiZ liver. Antibody against the
lysosomal membrane protein, LAMP-1, and the lysosomal hydrolase, cathepsin D, both of which are thought to be present within
autophagosomes, were used, followed by secondary anti-Ig antibody
bearing immunogold beads (3, 8, 17, 18, 24). The result in
Fig. 7C shows that the multilamellar, electron-dense
structures within PiZ mouse liver label positively for both of these proteins.
Quantitative morphometric analyses of the autophagosomes in the liver
cells at this steady state are shown in Fig.
8. The area of cytoplasm, not including
fat droplets, occupied by autophagosomes was determined in 10 hepatocytes from four different mice in each category. The results show
that there is a significant increase in autophagic vacuoles at steady
state mediated by fasting in the C57 black mouse, reaching 1.5% of the
area of the cytoplasm. Autophagosomes occupy 2.4% of the cytoplasm in
the PiZ mouse at baseline, with no significant change during fasting,
indicating that there is a marked constitutive increase in steady-state
autophagic vacuoles in the PiZ mouse and an inability to mount an
augmented autophagic response to fasting. Because there is no way to
separately determine the rate of formation of autophagosomes and the
rate at which autophagosomes fuse with lysosomes, it is not possible to
exclude the possibility that fasting causes an increase in both
formation and clearance of autophagosomes in the
1-AT-deficient liver in such a way that there is no net
change in steady-state levels of autophagosomes morphologically.

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Fig. 8.
Quantification of autophagic vacuoles in 3-mo-old PiZ and
C57 black mouse hepatocytes at baseline and after fasting. The percent
area of cytoplasm (excluding fat droplets) occupied by autophagic
vacuoles was determined from electron photomicrographs of 10 hepatocytes from each of 4 mice for each experimental condition shown.
Results are reported as means of the mice ± SD with
P < 0.05 between C57 and PiZ mice both at baseline and
after fasting by ANOVA.
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DISCUSSION |
In homozygous PIZZ
1-AT deficiency, a point
mutation confers polymerogenic properties on the mutant
1-ATZ molecule (2). This mutant protein is
retained in the ER rather than secreted. Most of the evidence in the
literature suggests that chronic liver injury and hepatocellular
carcinoma in some PIZZ individuals are the result of hepatotoxic
effects of ER
1-ATZ retention. However, it has been
difficult to explain why as many as 80-90% of PIZZ individuals
escape liver injury or have much milder liver injury detected
incidentally at autopsy. It has also been difficult to explain why the
liver disease can become apparent early in life in some patients and
then enter a honeymoon period compared with other patients in which
liver disease is only discovered much later. There have been very few
studies of potential genetic traits and/or environmental factors that
provide an explanation for this variability in liver disease phenotype
and in natural history. In one study using a genetic complementation
approach to express the mutant
1-ATZ protein in cell
lines from PIZZ individuals with or without known liver disease, we
found that inefficient degradation of
1-ATZ in the ER
correlated with susceptibility to liver disease (26).
However, it is still unknown whether inefficient ER degradation is
determined by genetic or environmental mechanisms, and there is no
information in the literature about how environmental factors may
interact with genetic determinants of susceptibility to liver disease.
To begin to examine these issues and the effect of environmental
conditions and developmental stage on susceptibility to liver injury in
1-AT deficiency, in this study we used the genetically engineered PiZ mouse as an in vivo model and fasting as a model environmental stressor. Fasting is a well-characterized stressor of
liver disease in infants and in experimental models of liver disease
and is particularly informative for
1-AT deficiency
because it is one of the best studied stimuli of the autophagic
response. Many previous studies have shown an increase in autophagic
activity in the liver during nutrient deprivation (3, 8, 9, 17, 18). Our recent morphological studies have shown that retention of mutant
1-ATZ in the ER is associated with an
autophagic response in cell culture and transgenic mouse model systems
as well as in human
1-AT-deficient liver
(24).
The results show that autophagy is constitutively activated, as
indicated by a steady-state increase in autophogic vacuoles, in the PiZ
mouse at baseline. Morphometric analysis suggests that the number of
autophagosomes in liver cells of the PiZ mouse at baseline is >50%
higher than that in liver cells of the C57 black mouse after
stimulation by fasting. In contrast to the C57 black mouse, fasting
does not lead to an increase in autophagosomes in the liver of the PiZ
mouse. These results therefore provide further evidence for the
integral relationship between the pathological effects of
1-AT deficiency and the autophagic response of the host.
If the autophagic response is intended to serve a protective role, by
clearing ER distended with aggregated mutant
1-ATZ
molecules or by suppressing tumorigenesis (7, 10), then
our data indicate that the
1-AT-deficient liver has
little reserve and would probably be easily overwhelmed by
physiological and pathological stressors. Furthermore, the consequences
of constitutive high-level activation of autophagy on the liver are
entirely unknown. From our search of the literature, the only other
condition in which there is accumulation of autophagic vacuoles under
homeostatic conditions is Danon disease (14, 22). In
contrast to
1-AT deficiency, however, autophagosomes
accumulate in Danon disease because of a genetic defect in the terminal
phases of autophagy, i.e., the fusion of autophagic vacuoles with
lysosomes and subsequent degradation within autolysosomes (14,
22). It should be noted, however, that currently available
methodology does not permit us to exclude the possibility that there is
no net change in steady-state levels of autophagosomes in the
1-AT-deficient liver during fasting because there are
increases in both formation and clearance of these vacuoles. The
results of the current studies indicate that the
1-AT-deficient liver is also susceptible to
physiological stress, as evidenced by increased mortality and
dysregulation of lipid metabolism. It is well known that hepatic
steatosis often follows fasting in wild-type mice, but there was a
massive increase in fat deposition in the fasting PiZ mouse noted in
this study. Recent studies have shown that there is increased hepatic
biogenesis, uptake of cholesterol and triglycerides, and hepatic
steatosis as a result of the increased sterol regulatory
element-binding protein signaling that accompanies the unfolded protein
response (16, 25). It is not uncommon to see hepatic
steatosis in patients with
1-AT deficiency being
evaluated for exacerbation of liver disease.
One of the other unexpected series of findings reported here is the
increase in size of
1-AT-containing globules during
aging and the increase in the number of
1-AT-containing
globules induced by fasting in the PiZ mouse at 2-6 mo of age.
Electron microscopic examination shows that these globules represent
portions of the ER because they are studded with ribosomes and
contiguous with normal-appearing ER. Moreover, these globules stain
positively for the resident ER proteins BiP and calnexin but not for
lysosomal enzymes. It is completely unknown at this time why the size
of these globules increases during aging and why the number of globules increases during fasting only in PiZ mice at the 2- to 6-mo period of
development. Immunofluorescent studies shown in Figs. 4 and 5 also show
that there is a change in distribution of
1-AT, BiP, and
calnexin exclusively in the globules after fasting. The disappearance of BiP and calnexin immunoreactivity from the diffuse reticular network
of the cytoplasm after fasting could not be attributed to a
disappearance of nondilated ER, because there was still abundant nondilated ER in liver cells evident by electron microscopy. Perhaps these globules represent specialized subdomains of the ER in which polymerized
1-ATZ, together with chaperones such as BiP
and calnexin, accumulates with an exceptionally long half-life. In
fact, the redistribution of BiP observed here during fasting bears a
remarkable resemblance to that described when ER-to-Golgi transport is
inhibited experimentally in yeast (13, 27) and attributed
to the need for chaperones to localize at ER exit sites where cargo
molecules or misfolded substrates accumulate.
 |
ACKNOWLEDGEMENTS |
Current address for D. Perlmutter: Dept. of Pediatrics, University
of Pittsburgh School of Medicine and Children's Hospital of
Pittsburgh, Pittsburgh, PA 15213.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
J. H. Teckman, Dept. of Pediatrics, Washington Univ. School
of Medicine, 660 S. Euclid Blvd., Campus Box 8208, St. Louis, MO 63110 (E-mail: teckman{at}pcfnotes1.wustl.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajpgi.00041.2002
Received 1 February 2002; accepted in final form 26 May 2002.
 |
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