Calorie-related rapid onset of alveolar loss, regeneration, and changes in mouse lung gene expression

Donald Massaro,1 Gloria DeCarlo Massaro,2 Alex Baras,3 Eric P. Hoffman,4 and Linda Biadasz Clerch2

Lung Biology Laboratory, Departments of 1Medicine, 2Pediatrics, and 3Biology, Georgetown University School of Medicine, Washington 20057-1481; and 4Center for Genetic Medicine, Children's National Medical Center, Washington, District of Columbia 20010

Submitted 17 September 2003 ; accepted in final form 28 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calorie restriction, followed by ad libitum refeeding, results, respectively, in loss and regeneration of pulmonary alveoli. We now show 35% of alveoli are lost within 72 h of onset of calorie restriction (2/3 decreased daily chow intake), and an additional 12% of alveoli are lost over a subsequent 12 days of calorie restriction. Tissue necrosis was not seen. Within 72 h of refeeding, after 15 days of calorie restriction, the number of alveoli returns to precalorie restriction values. Microarray lung gene profiling, in conjunction with Western and RNase protection assay, demonstrate an increase of granzyme and caspase gene expression 2–3 h after onset of calorie restriction. By 12 h, granzyme and caspase expression is no longer increased, but tumor necrosis factor death receptor expression is elevated. At 336 h, Fas death receptor expression is increased. Because granzymes are found only in cytotoxic lymphocytes (CTLs) and natural killer (NK) cells, we suggest calorie restriction activates these cells, initiating a series of molecular events that results in alveolar destruction. The evidence of involvement of CTLs and NK cells and the absence of necrosis are similar to alveolar destruction in chronic obstructive pulmonary disease.

apoptosis; caspases; cytotoxic lymphocytes; granzymes; microarray; natural killer T cells


THE REGULATION, molecular basis, and time required for the turnover (loss and formation) of pulmonary alveoli are poorly understood. Pulmonary emphysema, an important component of chronic obstructive pulmonary disease (COPD), is characterized by apoptosis of alveolar wall cells (2), little or no necrosis (6), and, surprisingly, an all-or-nothing (68, 71) progressive destruction of individual alveoli (26, 56, 67). These changes are thought to be due to insufficient intra-alveolar antiprotease activity, inflammation, and oxidant stress (3, 6, 16, 26, 55). Experiments on animals and humans with emphysema suggest proteases and oxygen radicals released by neutrophils, lymphocytes, and macrophages play a key role in alveolar destruction (6, 11, 24, 27, 50, 63). However, therapies of COPD based on these notions of pathogenesis have been notably unsuccessful in slowing the rate of loss of diffusing capacity, as has cessation of cigarette smoking (56, 67). Furthermore, a means of inducing alveolar regeneration in COPD has not been established.

Calorie restriction of adult rodents, followed by ad libitum access to food, activates, respectively, endogenous programs of alveolar destruction (23, 30, 32, 52) and of alveolar regeneration (33, 39, 52). Starvation in adult humans leads to emphysema-like changes in the lung (9, 12, 73), suggesting the endogenous program(s) of alveolar destruction is conserved in humans. We now show in adult male mice that calorie restriction results, within 2 h, in molecular changes in lung consistent with activation of pathways of apoptosis; by 72 h of calorie restriction, alveolar loss has occurred. Ad libitum refeeding after 15 days of calorie restriction results in alveolar regeneration within 72 h. Some of the changes of gene expression during calorie restriction-induced alveolar destruction are similar to those found in COPD (3, 6, 11).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and experimental manipulations. Adult male C57BL/6J mice were purchased from Jackson Laboratory. Upon arrival at Georgetown, they were housed three to four per cage in the Department of Comparative Medicine on a 12:12-h light-dark schedule and were allowed ad libitum Ralston Purina Laboratory Chow 5001. After at least 1 wk at Georgetown, the mice were housed one per cage, and the amount of chow eaten each day was measured. On the basis of the average daily weight of chow eaten over 4–5 days by each mouse, we diminished the daily allotment of chow provided to some mice by 2/3 (33). After being calorie restricted, some mice were allowed ad libitum access to food; other mice were always allowed ad libitum access to food. All mice were always provided tap water ad libitum.

The average daily food consumption in mice fed ad libitum was ~4.2 g/day. Diminishing daily Ralston Purina Laboratory Chow 5001 intake by 2/3 provides 30 IU of retinol each day. The minimum daily requirement of retinol for mice is 1–2 IU (61). Therefore, this study is not influenced by retinol deficiency, which requires months of a retinol-free diet to produce (61), but can alter alveolar structure (4). We killed animals by cutting large vessels in the abdomen after establishing a surgical level of anesthesia with xylazine (~10 mg/kg) plus ketamine (~75 mg/kg). All procedures were approved by the Georgetown University Animal Care and Use Committee and comply with the United States Department of Agriculture and National Institutes of Health guidelines.

Morphological and morphometric studies. Mice were anesthetized with xylazine plus ketamine. The trachea was intubated, the diaphragm punctured, and cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, was infused into the trachea at a transpulmonary pressure of 20 cmH2O. The trachea was ligated, and the lungs were removed from the thorax. Fixation was continued at 0–4°C for 2 h. Lung volume was measured by volume displacement (54). Lungs were cut into blocks. Blocks were selected for further processing using a systematic sampling technique (14), washed in cacodylate buffer, postfixed for 1 h at 4°C in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated, and embedded in epoxy resin (38).

We cut serial sections at ~0.8-µm thickness and sectioned three blocks per animal. Each group of serial sections was cut to a depth of 150–250 µm. To identify alveolar air spaces, gas-exchange structures were followed through a complete set of prints of serially sectioned lung (38, 39). An alveolus was defined as a structure with a mouth that communicated with a common air space; the latter was designated an alveolar duct.

The selector method (13) was used to choose alveoli for analysis (38, 39). This method allows alveoli to be selected for measurement based on number, uninfluenced by size, shape, or orientation. Thirty alveoli were analyzed per mouse. The volume of an alveolus was estimated by the point-sampled intercepts method (22), and the number of alveoli was calculated as previously described (38).

To determine alveolar surface area, sections ~0.8 µm thick were cut from each of 10 tissue blocks, which provided 10 sections per mouse, and stained with toluidine blue. Lung sections were photographed using a Reichert Microstar IV microscope and Polaroid film 667; final prints were at a magnification of x160. Point and intersection counting was used to determine alveolar surface area (70). Volume density was estimated by counting the number of points that fell on the object under analysis (alveolar tissue, gas-exchange air space, or conducting structures) divided by the total number of test points (70). All measurements were made without knowledge of the manipulation to which the mice had been subjected.

RNase protection assay. RNase protection assay (RPA) analysis was performed with probes to caspase 2, 3, 6, 8, and 12 according to the manufacturer's instructions (mAPO-1 multiprobe mouse apoptosis-1 template set from Pharmingen). The probes were labeled by in vitro transcription using RiboQUANT (BD Biosciences). We used the concentration of ribosomal protein L32 as an internal standard to which the concentration of the caspase mRNAs were related. The concentration of L32 was not different between 48-h ad libitum-fed mice (3,681 ± 308 densitometry units) and 48-h calorie-restricted mice (3,530 ± 237 densitometry units).

Expression profiling. The right and left lung from each mouse were placed in a separate piece of aluminum foil with lungs from four other mice on the same diet, i.e., food ad libitum or calorie restricted (Fig. 1). Each package of aluminum foil had right and left lungs from two (2- to 96-h time points) or five (336-h time point) different mice. All the lungs in each foil package were disrupted to form one homogenate representing each package. Total RNA was separately extracted from each homogenate using TRIzol reagent (GIBCO BRL) and purified further using RNeasy (Qiagen). The RNA was converted into double-stranded cDNA using SuperScript Choice System (GIBCO BRL) with an oligo(dT) primer containing T7 RNA polymerase promoter (Genset). Double-stranded cDNA was purified by phenol/chloroform extraction and then used for in vitro transcription with an ENZO BioArray RNA transcript-labeled kit. Biotin-labeled cRNA was purified by RNeasy (Qiagen) and randomly fragmented before hybridization to an Affymetrix Murine MG-U74Av2 GeneChip using an Affymetrix Fluidics Station 400 and a Hewlett Packard G2500A Gene Array scanner. Expression profiling data can be found at NCBI GEO (http://www.ncbi.nlm.nih.gov/geo), accession series GDS241 (Alveoli destruction time course). Supplementary Table S3 is available online at the AJP–Lung Cellular and Molecular Physiology web site.1



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1. Experimental design for lung gene profiling experiments. Some mice were always allowed chow ad libitum; others were calorie restricted to one-third of their individual average chow intake over the proceeding 4–5 days. All mice were always allowed tap water ad libitum. The designation "lungs of 5 mice = 1 N" indicates the lungs of 5 mice were separated into right and left lung, and 3 right and 2 left lungs were combined and total RNA isolated; separately, 3 left and 2 right lungs were also combined and total RNA isolated. Both samples of total RNA were separately used to generate biotin-labeled cRNA. Each sample of the latter was hybridized on separate Affymetrix chips, Murine MG-U74Av2, which are referred to as replicate 1 and replicate 2. We calculated the mean expression of the 2 chips for each gene. For each time, there was an N of 2 for calorie restriction (CR) and for ad libitum-fed mice, except for the 336-h experiment where the N was 6 for CR and 4 for ad libitum-fed mice. ss, Single stranded; ds, double stranded.

 

Analysis of expression profiling. We used an experimental design that attempts to account for potential diurnal variation in gene expression (Fig. 1). GeneSpring 5.1 (Silcon Genetics) software was used to analyze the data. Because all RNA analyses were performed on duplicate Affymetrix gene chips, the GeneSpring global error model (http://www.silicongenetics.com) was used to estimate variations between chips and between treatments except for the results from the 336-h experiments. On the latter, an unpaired, two-tailed t-test analysis was used to determine the statistical significance of differences of gene expression between ad libitum-fed and calorie-restricted mice (59).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body mass changed rapidly with altered food intake. Body mass fell 10% within 12 h of onset of calorie restriction (Table 1). Two-thirds of the fall that took place during 15 days of calorie restriction occurred within 72 h of initiating calorie restriction. By 12 h of refeeding, after 15 days of calorie restriction, body mass increased 28%. Body mass was not different among groups of mice always allowed chow ad libitum (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Changes in body mass in response to calorie restriction by 2/3 and to ad libitum refeeding after calorie restriction

 

Morphology of gas-exchange tissue during altered food intake. We did not detect evidence of tissue necrosis (Fig. 2). Alveolar walls appeared thinner and alveoli larger in calorie-restricted mice compared with ad libitum and with calorie-restricted ad libitum-refed mice.



View larger version (70K):
[in this window]
[in a new window]
 
Fig. 2. Morphological response to CR and calorie restriction-refeeding (CR-RF). Mice were fed ad libitum for 3 (A) or 15 (C) days; other mice were calorie restricted for 3 (B) or 15 (D) days; some of the latter were refed ad libitum for 3 days (E). Bar scale = 50 µm.

 

Calorie restriction caused a fall in the volume of gas-exchange tissue; ad libitum refeeding raised it. Neither total lung volume (Fig. 3A) nor gas-exchange air volume (Fig. 3B) differed between ad libitum-fed and calorie-restricted mice. However, the volume of gas-exchange tissue was 30% lower after 15 days of calorie restriction than in ad libitum-fed mice; this difference was not present by 72 h of ad libitum refeeding (Fig. 3C).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Lung and gas-exchange air and tissue volume. A–C: mice were fed ad libitum, calorie restricted, or calorie-restricted and then refed ad libitum for the times shown. Means ± SE are given except for fed (F) 3 days in B and C. Numerals within the bars indicate the number of mice. *P <= 0.029 vs. same day fed ad libitum; **P = 0.009 vs. 15 days calorie restricted.

 

Alveolar loss was detected within 72 h of onset of calorie restriction and regeneration within 72 h of ad libitum refeeding. The volume of individual alveoli increased 44% within 72 h of calorie restriction (Fig. 4A). This represented ~75% of the increase of the volume of individual alveoli that occurred over the entire 15 days of calorie restriction. The number of alveoli exhibited a correspondingly rapid decline (Fig. 4B). Alveolar surface area also fell within 72 h of calorie restriction (Fig. 4C). By 72 h of ad libitum refeeding, after 15 days of calorie restriction, the volume of individual alveoli and the number of alveoli had returned to values present in mice never calorie restricted. We did not detect a statistically significant increase of alveolar surface area after ad libitum refeeding. This failure may reflect the fact that area changes to the power 2, volume to the power 3, and hence area changes are only 2/3 as large as those of volume.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Average volume of individual alveoli (A), alveolar number (B; Na), and alveolar surface area (C; Sa). Mice were allowed food as in Fig. 3. Means ± SE are given. Numerals within the bars indicate the number of mice. *P < 0.008 vs. ad libitum fed; **P < 0.005 vs. 15-day (D) calorie restriction (CR); ***P < 0.007 vs. ad libitum fed and vs. CR-RF; {triangleup}P < 0.01 vs. ad libitum fed and <0.03 vs. CR-RF. {blacktriangleup}P < 0.05 vs. all others.

 

Comparison of gene expression from the same lungs on duplicate chips. A scatter plot analysis of mRNA from mice allowed food ad libitum or that were calorie restricted for 15 days, respectively, revealed 93.4 ± 0.2 and 93.4 ± 0.3 of the intensity of points fell within two standard deviations of the line of identity (Fig. 5). As expected, there was greater variation between duplicate chips at low levels of expression than at high levels (Fig. 5). The greater variance impairs detection of intergroup differences among genes expressed at low levels.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. Scatter plot of replicate expression values. Each point represents duplicate measurements of the same gene. If replicate expression values were identical, all points would fall on the diagonal line of identity. In the ad libitum-fed mice, 93.4 ± 0.2% of the replicates fell within 2 SD of the line of identity (A); for calorie-restricted mice, 93.4 ± 0.3% of the replicates fell within 2 SD of the line of identity (B).

 

Analysis of lung gene expression during calorie restriction. The expression of all genes and expression sequence tags (ESTs) queried in our experiments may be obtained from the database "Public Expression Profile Resource" (http://microarray.cnmcresearch.org/pgadatatable.asp). Because of the massive amount of data generated by microarray gene profiling, we have focused on only two categories of genes as identified by GeneSpring ontology software. We selected genes involved in cell death or proteolysis (Supplementary Table S3, Figs. 6 and 7) because these biological processes are considered important in experimental cigarette smoke-produced emphysema, emphysema in human COPD, and in lung calorie-related apoptosis, increased proteolysis, and alveolar loss (2, 3, 6, 11, 24, 27, 39, 64). However, many other cellular changes, e.g., related to glucose and lipid metabolism (5, 21, 64), take place in response to calorie restriction and may be reflected in the gene expression results shown in Supplementary Table S3 and Figs. 6 and 7. Furthermore, we have shown only genes whose expression in lungs of calorie-restricted mice compared with lungs of ad libitum-fed mice had a P value <=0.05 by the GeneSpring global error model or by an unpaired, two-tailed t-test. With these restrictions, 120 genes were higher in lungs of calorie-restricted mice, and 137 were lower compared with lungs of ad libitum-fed mice.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 6. Temporal expression of genes related to apoptosis. Hierarchical clustering was used to order the genes. Blue indicates downregulation of genes in CR mice compared with the same genes in ad libitum-fed mice at the same time. Red indicates upregulation of genes from CR mice compared with the same gene from ad libitum-fed mice at the same time. The intensity of the color directly reflects the magnitude of increase or decrease of gene expression.

 


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 7. Temporal expression of genes related to proteolysis. The experimental manipulations, clustering, and color code are as described in Fig. 6.

 

Within 2 h of onset of calorie restriction, the mRNA of a TNF ligand (TWEEK) and of granzyme A, an IL-1{beta} converting enzyme (24), was elevated in lungs of calorie-restricted mice (Supplementary Table S3). By 3 h, granzyme B protein concentration, which was determined by Western analysis, was increased in lungs of calorie-restricted mice (vide infra). The 2- and 4-h mRNA values for caspase 3, considered individually, did not achieve statistically significant differences among treatment groups. However, when the 2- and 4-h calorie restriction results were combined (n = 4), and the 2- and 4-h ad libitumfed results were combined (n = 4), caspase 3 mRNA was 1.3-fold higher in lungs of calorie-restricted mice (P = 0.038). Four hours after institution of calorie restriction, the mRNA of kallikrein, caspase 14, TNF receptor superfamily member 17, and an aryl hydrocarbon receptor, which activates proteosomes, were higher in lungs of calorie-restricted mice than in lungs of ad libitum-fed mice (Supplementary Table S3). These findings are consistent with increased serine protease activity (kallikrein), diminished cell-extracellular matrix interaction (caspase 14) (46), and increased signaling via TNF ligand (TWEEK) and TNF receptors (TNF receptor superfamily member 17). By contrast, at the same time, a RING finger mRNA, which targets proteins for proteasomal destruction (17) and programmed cell death 8, an apoptosis-inducing factor (62), were diminished in lungs of calorie-restricted mice (Supplementary Table S3).

These findings suggest the early onset of four routes to the previously demonstrated (39) alveolar wall cell death: 1) TNF mediated, 2) destruction of extracellular matrix (caspase 14), 3) caspase-independent cell death (granzymes A and B) (36), and 4) caspase-dependent cell death (caspase 3 and granzyme B) (36). Granzymes A and B are serine protease activators of cell death produced only by CTLs and NK cells and are stored in secretory granules (36). CTLs require 20–24 h to become activated and form granules (36). NK cells in the steady state bear granules containing granzymes and other cytotoxicity effectors that can be released instantaneously (36). Therefore, it is likely NK cells are the initial source of cell-destroying granzymes after the onset of calorie restriction.

By 12 h of calorie restriction, evidence of DNA damage was indicated by growth arrest and DNA damage-inducible 45{gamma} (Gadd 45{gamma}) mRNA being higher in calorie-restricted mice than ad libitum-fed mice (Supplementary Table S3). The main role of Gadd genes is to block proliferation at G1 and G2 check points in response to DNA damage (33, 74). The mRNAs of casinolytic protease X and of ubiquitin-specific protease 2, which are involved in destruction of cellular proteins (20, 33), were also elevated in lungs of calorie-restricted mice (20, 33). In contrast to the molecular effects of calorie restriction that favor matrix destruction and apoptosis within the first 12 h, the higher concentration of Bcl-2, which interrupts an apoptosis cascade (49), the lower concentrations of the mRNA of three ADAM (a disintegrin and metalloprotease) family proteins (ADAM 7, 17, and 28), and of programmed cell death 8 mRNA do not favor apoptosis (10, 41, 58).

The simultaneous presence of molecular changes favoring and opposing apoptosis may reflect the lung's cellular heterogeneity, some cells being expendable, others not. In this regard, endophilin, which is involved in endocytic removal of plasma membrane receptors (43), was elevated in calorie-restricted mice and thereby could determine which alveolar wall cells respond to the nonspecific presentation of extracellular signals to all alveolar wall cells. It is also important to acknowledge these changes of gene expression are at the level of mRNA, whereas the functions of the genes are effected by proteins.

After the first 4 h, the mRNAs of granzyme A and of granzyme B either were not different between lungs of calorie-restricted and ad libitum-fed mice or were lower in the former than the latter (Supplementary Table S3). However, evidence consistent with cell death mediated by TNF receptors (increased mRNAs of Tnfrsf 1a and of TRAF 2) persisted through 336 h (Supplementary Table S3). The mRNA of an ADAM family member (ADAM 23) was higher in lungs of calorie-restricted mice than in lungs of ad libitum-fed mice (Supplementary Table S3). This is relevant to the increase of TNF receptors because ADAM family proteases cause release of soluble TNF (41), which can induce apoptosis (48). The mRNA of angiotensin-converting enzyme, which is formed in endothelial cells and whose protein is required for TNF-{alpha}-induced apoptosis of alveolar epithelial cells (69), was elevated at 24, 48, and 96 h. Furthermore, the transcription factor CCAAT/enhancer-binding protein, which is induced by stress (19) and whose activation by IL-12 may induce expression of TNF-{alpha} (29), was increased at 72, 96, and 336 h. The mRNA of tissue plasminogen activator, whose protein converts plasminogen, an inactive serine protease, to plasmin, an active serine protease, was elevated at 24 and 96 h. Granzyme C mRNA was elevated 2.4-fold at 96 h in lungs of calorie-restricted mice (Supplementary Table S3). Granzyme C, as granzyme A, can produce caspase-independent cell death but is also a powerful cause of cell death by pathways different from those used by granzymes A and B (36). By 336 h, Fas-associated factor mRNA was elevated in lungs of calorie-restricted mice, suggesting the Fas ligand death pathway had been activated. However, and consistent with the morphometric results (Figs. 3 and 4), the gene expression results at 336 h indicate apoptosis had diminished but that extracellular matrix remodeling persisted. These findings are consistent with the substantial apoptosis (39), alveolar wall thinning (Fig. 3C), and alveolar loss (Fig. 4B) that occur over the first 72 h of calorie restriction and continues, albeit at a slower pace, over the full 336 h of calorie restriction. There were also changes of gene expression consistent with, and explanatory of, the previously described twofold increase of proteolysis in the lung during calorie restriction (64). For example, the mRNA of calpain 2, which is a Ca2+-activated intracellular cysteine protease (45), was elevated at 24, 48, and 336 h (Supplementary Table S3).

To verify some of the intergroup differences in gene expression identified by use of microarray, Western blot and RPA analysis were performed. The concentration of granzyme B (densitometry units/milligram lung protein) in lungs of calorie-restricted mice, 3 h after the onset of calorie restriction, was (means ± SE) 8.5 ± 0.2 (n = 4), and in lungs of mice fed ad libitum and killed at the same time, it was 7.7 ± 0.1 (n = 3, P = <0.02). We also compared the microarray assessment of the mRNA of some caspases with the expression of the same mRNAs determined by RPA analysis. The RPA analysis confirmed the array analysis for all five genes tested (Table 2). However, it is important to point out that, except for granzyme B, all other changes were at the RNA level, and the translation of these changes to the protein level is needed to more completely understand gene expression in the lung during calorie restriction.


View this table:
[in this window]
[in a new window]
 
Table 2. Comparison of lung gene expression assessed by microarray and RPA analysis

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental design for microarray. We believe our approach to the microarray studies has been stringent. The use of lungs from two or five mice for each n value should have diminished the impact of interanimal variability. Comparing calorie-restricted to ad libitum-fed mice killed at the same time in the 24-h cycle limited the potentially confounding factor of diurnal variation in gene expression. Duplicate processing, i.e., separate isolation of RNA and hybridization of RNA from an equal mix of right and left lungs on duplicate chips, provided two sets of data and is expected to control for differences in RNA isolation, biotinylation of RNA, and variability in hybridization to the GeneChip. The complete agreement, for the genes tested, of the array analysis with Western blot and RPA supports the validity of the methods and increases confidence in the expression results we did not try to confirm. Considering only genes that exhibited statistically significant intergroup differences further increases confidence in the results of the microarray experiments.

Calorie restriction. Periods of food scarcity in air-breathing organisms must have occurred since the Devonian when lung fish estivated during periods of drought. To survive such periods of not eating, organisms produce substrate, by destruction of comparatively nonessential structures, for gluconeogenesis to provide glucose for the brain and amino acids to maintain muscle. For example, among marine iguanas, those that shrink long bones to the greatest extent during food scarcity survive the longest (72). With respect to the lung, its only essential function is to provide sufficient gas-exchange surface to meet the organism's need for oxygen. Therefore, some lung tissue would be expendable as total organismal (42) and lung oxygen consumption (21) fall during calorie restriction. The lung's respiratory quotient decreases during calorie restriction (21), indicating use of lipid rather than glucose, the lungs preferred substrate in ad libitum-fed animals (26). Studies with 2-deoxy-D-glucose, which is not metabolized beyond being phosphorylated, reveal the diminished utilization of glucose during calorie restriction is at a step(s) beyond phosphorylation of glucose (5). In addition to decreased utilization of glucose, calorie restriction doubles the rate of proteolysis in the lung (64). Therefore, in the lung, calorie restriction results in metabolism of lipid in preference to glucose and in substantial proteolysis; the former would increase glucose available for use by the brain, and the latter would provide amino acids for gluconeogenesis and to maintain muscle. Refeeding elevates O2 consumption (42), thereby increasing the need for alveolar surface area, and lung regeneration rapidly takes place.

Alveolar destruction. At each time queried, some intergroup differences of gene expression favored cell death and tissue destruction, others cell survival. This suggests that, in a cellularly heterogeneous organ, certain cell types are selected for destruction, others for survival. As a whole, the results of our gene expression studies are consistent with the following during calorie restriction: 1) NK cells, activated by as yet unknown stimuli, initiate the molecular changes responsible for alveolar destruction; 2) degradation of extracellular matrix is a key initiating event in alveolar wall cell apoptosis and alveolar destruction; and 3) apoptosis of alveolar wall cells initially occurs by granzyme-induced, TNF receptor, and ADAM family pathways, later by Fas pathways.

The cytotoxicity of CTL and NK cells is effected by their secretion of proteases, e.g., granzymes, present in secretory granules (48). Granzymes are serine proteases produced only by NK cells and by activated CTLs (48). However, naive CTLs do not contain preformed granzymes; their formation, following CTL activation, takes 1–3 days (48). By contrast, NK cells have preformed granules that contain granzymes that can be released within minutes after the NK cell receives an appropriate signal (48). Therefore, NK cells are probably responsible for molecular evidence of cell death and alveolar destruction that is present very shortly after initiation of calorie restriction. The rapid onset, in a nocturnal mammal, of changes in gene expression after institution of calorie restriction during day-light, is consistent with the frequent episodes of food consumption in mice even during daytime (34).

Granzyme A, whose mRNA is elevated 2 h after onset of calorie restriction, does not play a primary role in apoptosis but does destroy extracellular matrix proteins (36, 48). This supports the notion that an early key event in apoptosis of alveolar wall cells may be disruption of the extracellular microenvironment, which initiates cell death by apoptosis. Granzyme B can also cause apoptosis by altering the extracellular environment but can more directly initiate an intracellular cascade that leads to apoptosis (36). The ADAM family of serine proteases regulates cell behavior by modifying the extracellular matrix (50); it also causes the release of soluble TNF, which can initiate apoptosis (34, 40). The increase of ADAM mRNA and of TNF receptor mRNA after 12 h of calorie restriction, in view of the ability of ADAM proteins to release soluble TNF, suggests a common upstream regulator of ADAM and TNF receptor genes.

Alveolar loss with, or without, a change of lung tissue elastic recoil. It is becoming apparent that alveolar destruction can occur with, or without, loss of lung tissue elastic recoil. For example, alveolar destruction in humans with COPD (8) and alveolar destruction produced by the instillation of elastase (37) or active caspase 3 (2) are associated with a decrease of lung elastic tissue recoil. By contrast, calorie restriction and corticosteroids both cause alveolar loss (7, 39), but without a loss of lung recoil (15, 18, 51). Alveolar loss in COPD, and that produced by exogenous elastase, continues even after the inciting agent has been removed, e.g., cessation of cigarette smoking in COPD (56, 67) and absence of elevated elastase activity in experimental exogenous elastase-induced emphysema (37). The number of alveoli lost between 3 days and 15 days of calorie restriction is not different (Fig. 4). These considerations suggest alveolar loss associated with loss of elastic tissue recoil is not, or cannot be, endogenously regulated; alveolar loss without loss of elastic tissue recoil is endogenously regulated. Because apoptosis of alveolar wall cells occurs in all these forms of alveolar wall destruction (2, 6, 39), the apoptosis does not seem to drive the process unless, and this remains to be seen, apoptosis of specific lung cells, e.g., alveolar capillary endothelial cells (31), is determinative.

Potential clinical relevance. Although alveolar destruction occurs in a general population as part of biological aging (60, 65), the most widely recognized and medically important cause of alveolar destruction is COPD (3, 6, 26), for which cigarette smoking is the major risk factor (25, 57). The main current theories of the pathogenesis of COPD are intra-alveolar protease-antiprotease imbalance, chronic inflammation, oxidant damage, and an interaction among these (3, 6, 16, 26, 55). Smoking cessation slows the loss of conducting airway function (1, 44). However, therapies aimed at the currently considered etiological basis of alveolar destruction (3, 16), including remarkably, even smoking cessation (53, 56, 67), fail to slow the loss of gas-exchange function in individuals with COPD.

In addition to polymorphonuclear leukocytes (26) and macrophages (24, 27, 63), CTL and NK cells, via their secretory products, which include granzymes and TNF, are thought to play a role in alveolar destruction in emphysema (3, 11, 50). Our findings demonstrating the remarkably early onset of increased expression of granzymes, caspases, and TNF ligands and receptors strongly suggest these molecules are important to alveolar destruction in calorie-related alveolar loss, as they are thought to be in COPD (3, 11, 50).

The evidence of involvement of granzymes, which are serine proteases, in calorie restriction-related alveolar loss, the likelihood calorie restriction-related alveolar loss is highly conserved from "mouse to man" (9, 10, 12, 23, 30, 39, 52, 73), and the failure of endogenous serine antiproteases to control granzyme activity in the inflamed lung (66) suggest these properties of lung have been conserved because they allow lung destruction during food scarcity, thereby providing a survival advantage. The evidence that CTL, NK cells, granzymes (40), and TNF play an important role in alveolar destruction in COPD (11, 40, 50) and in calorie restriction-related alveolar loss raises the possibility that COPD and calorie restriction share the same molecular signals for alveolar loss. The fact that alveolar loss slows as the duration of calorie restriction increases and is reversed with ad libitum refeeding indicates there are endogenous regulators of the process of alveolar destruction and regeneration. We raise the possibility that in COPD, even in the absence of calorie restriction, the pathways of calorie restriction-related alveolar destruction are inappropriately activated, and endogenous controls fail, or the inappropriate stimulus for alveolar destruction persists, accounting for the, so far, irremediable, progressive loss of gas-exchange function (56, 67).


    ACKNOWLEDGMENTS
 
We thank Zofia Opalka, Valerie Tyner, and Megan Ferringer for technical assistance. D. Massaro is Cohen Professor of Medicine. A. Baras did this work while a Howard Hughes Scholar of the Department of Biology at Georgetown University.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-37666, HL-20366, HL-47413, and HL-66614-01 HOPGENE.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. Massaro, Lung Biology Laboratory, Box 571481, Preclinical Science Bldg., GM-12, Georgetown Univ. School of Medicine, 3900 Reservoir Road, NW, Washington, DC 20057-1481 (E-mail: massarod{at}georgetown.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.

1 The Supplementary material for this article (Supplementary Table S3) is available online at http://ajplung.physiology.org/cgi/content/full/00333.2003/DC1. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anthonisen NR, Connett JE, Kiley JP, Altose MD, Bailey WC, Buist AS, Conway WA Jr, Enright PL, Kanner RE, O'Hara P, Owens GR, Scanlon PD, Tashkin DP, and Wise RA. Effect of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV1. The Lung Health Study. JAMA 272: 1497-1505, 1994.[Abstract]
  2. Aoshiba K, Yokohori N, and Nagai A. Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Biol 28: 555-562, 2003.[Abstract/Free Full Text]
  3. Barnes PJ. New concepts in chronic obstructive pulmonary disease. Annu Rev Med 54: 113-129, 2003.[CrossRef][Medline]
  4. Baybutt RC, Hu L, and Molteni A. Vitamin A deficiency injures lung and liver parenchyma and impairs function of rat type II pneumocytes. J Nutr 130: 1159-1165, 2000.[Abstract/Free Full Text]
  5. Chaisson CF and Massaro D. 2-Deoxy-D-glucose uptake by lung slices from fed and fasted rats. J Appl Physiol 44: 380-383, 1978.[Abstract/Free Full Text]
  6. Chapman HA Jr and Shi GP. Protease injury in the development of COPD: Thomas A. Neff Lecture. Chest 117: 295S-299S, 2000.[Free Full Text]
  7. Choe KH, Taraseviciene-Stewart L, Scerbavicius R, Gera L, Tuder RM, and Voelkel NF. Methyprednisolone causes matrix metalloproteinase-dependent emphysema in adult rats. Am J Respir Crit Care Med 167: 1516-1521, 2003.[Abstract/Free Full Text]
  8. Christie RV. The elastic properties of the emphysematous lung and their significance. J Clin Invest 13: 295-321, 1934.
  9. Cook VJ, Coxson HO, Mason AG, and Bai TR. Bullae, bronchiectasis and nutritional emphysema in severe anorexia nervosa. Can Respir J 8: 361-365, 2001.[Medline]
  10. Cornwall GA and Hsia N. ADAM 7, a member of the ADAM (a disintegrin and metalloprotease) gene family is specifically expressed in the mouse anterior pituitary and epididymis. Endocrinology 138: 4262-4272, 1997.[Abstract/Free Full Text]
  11. Cosio MG, Majo J, and Cosio MG. Inflammation of the airways and lung parenchyma in COPD: role of T cells. Chest 121: 160S-165S, 2002.[Abstract/Free Full Text]
  12. Crow S, Praus B, and Thuras P. Mortality from eating disorders–a 5- to 10-year record linkage study. Int J Eat Disord 26: 97-101, 1999.[CrossRef][ISI][Medline]
  13. Cruz-Orive LM. Particle number can be estimated using a disector of unknown thickness: the selector. J Microsc 145: 121-142, 1987.[ISI][Medline]
  14. Cruz-Orive LM and Weibel ER. Sampling designs for stereology. J Microsc 122: 235-257, 1981.[ISI][Medline]
  15. D'Amour R, Clerch L, and Massaro D. Food deprivation and surfactant in adult rats. J Appl Physiol 55: 1413-1417, 1983.[Abstract/Free Full Text]
  16. Dickson R and Strange C. DLCO in the NHLBI {alpha}1-antitrypsin deficiency registry (Abstract). Am J Respir Crit Care Med 165: A591, 2002.
  17. Fang S, Lorick KL, Jensen JP, and Weissman GP. RING finger ubiquitin protein ligases: implications for tumorogenesis, metastasis and for molecular targets in cancer. Semin Cancer Biol 13: 5-14, 2003.[CrossRef][ISI][Medline]
  18. Gail DG, Massaro GD, and Massaro D. Influence of fasting on the lung. J Appl Physiol 42: 88-92, 1977.[Abstract/Free Full Text]
  19. Garcia-Montero A, Vasseur S, Mallo GV, Soubeyran P, Dagorn JC, and Jovanna IL. Expression of the stress-induced p8 mRNA is transiently activated after culture medium change. Eur J Cell Biol 80: 720-725, 2001.[ISI][Medline]
  20. Gong L, Kamitani T, Millas S, and Yeh ET. Identification of a novel isopeptidase with dual specificity for ubiqiutin- and NEDD8-conjugated proteins. J Biol Chem 275: 14212-14216, 2000.[Abstract/Free Full Text]
  21. Gregorio CA, Gail DB, and Massaro D. Influence of fasting on lung oxygen consumption and respiratory quotient. Am J Physiol 230: 291-294, 1976.[Abstract/Free Full Text]
  22. Gundersen HJG and Jensen EB. Stereological estimate of the volume-weighted mean volume of arbitrary particles observed on random samples. J Microsc 138: 127-142, 1995.
  23. Harkema JR, Mauderly JL, Gregory RE, and Pickrell JA. A comparison of starvation and elastase models of emphysema in the rat. Am Rev Respir Dis 129: 584-591, 1984.[ISI][Medline]
  24. Hautamake RD, Kobayashi DK, Senior RM, and Shapiro SD. Macrophage elastase is required for cigarette smoke-induced emphysema. Science 277: 2002-2004, 1997.[Abstract/Free Full Text]
  25. Higgins M. Risk factors associated with chronic obstructive pulmonary disease. Ann NY Acad Sci 624: 7-17, 1991.[Abstract]
  26. Hogg JC and Senior RM. Chronic obstructive pulmonary disease–Part 2: pathology and biochemistry of emphysema. Thorax 57: 830-834, 2002.[Abstract/Free Full Text]
  27. Hunninghake GW, Davidson JM, Rennard S, Szapiel S, Gadek JE, and Crystal RG. Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science 212: 925-927, 1981.[ISI][Medline]
  28. Irmler M, Hertig S, MacDonald HR, Sadoul R, Becherer JD, Proudfoot A, Solari R, and Tschopp J. Granzyme A is an interleukin 1{beta}-converting enzyme. J Exp Med 181: 1917-1922, 1995.[Abstract]
  29. Jana M, Dasqupta S, Saha RN, Liu X, and Pahan K. Induction of tumor necrosis factor (TNF-{alpha}) by interleukin-12p40 monomer and homodimer in microglia and macrophages. J Neurochem 86: 519-528, 2003.[CrossRef][ISI][Medline]
  30. Karlinsky JB, Goldstein RH, Ojserkis B, and Snider GL. Lung mechanics and connective tissue levels in starvation-induced emphysema in hamsters. Am J Physiol Regul Integr Comp Physiol 251: R282-R288, 1986.[Abstract/Free Full Text]
  31. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, and Voelkel NF. Inhibition of VEGF receptors causes lung cell apopotosis and emphysema. J Clin Invest 106: 1311-1319, 2000.[Abstract/Free Full Text]
  32. Kerr JS, Riley DJ, Lanza-Jacoby S, Berg RA, Spilker HC, Yu SY, and Edelman NH. Nutritional emphysema in the rat. Influence of protein depletion and impaired lung growth. Am Rev Respir Dis 131: 644-650, 1985.[ISI][Medline]
  33. Korabiowska M, Cordon-Cardo C, Betke H, Schlott T, Kotthaus M, Stachura J, and Brinck U. GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis. Histol Histopath 17: 805-811, 2002.[ISI][Medline]
  34. Kurokawa M, Akino K, and Kanda K. A new apparatus for studying feeding and drinking in the mouse. Physiol Behavior 70: 105-112, 2000.[CrossRef][ISI][Medline]
  35. Lareu RR, Lacher MD, Bradley CK, Sridaran R, Friis RR, and Dharmarajan AM. Regulated expression of inhibitor of apoptosis protein 3 in the rat corpus luteum. Biol Reprod 68: 2232-2240, 2003.[Abstract/Free Full Text]
  36. Lieberman J. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 3: 361-370, 2003.[CrossRef][ISI][Medline]
  37. Lucey EC, Stone PJ, Christensen TG, Brewer R, and Snider GL. An 18-month study of the effects on hamster lungs of an intratracheally administered human neutrophil elastase. Exp Lung Res 14: 671-686, 1988.[ISI][Medline]
  38. Massaro GD and Massaro D. Formation of pulmonary alveoli in rats: postnatal effect of prenatal dexamethasone. Am J Physiol Lung Cell Mol Physiol 263: L37-L41, 1992.[Abstract/Free Full Text]
  39. Massaro GD, Radaeva S, Clerch LB, and Massaro D. Lung alveoli: endogenous programmed destruction and regeneration. Am J Physiol Lung Cell Mol Physiol 283: L305-L309, 2002.[Abstract/Free Full Text]
  40. Möllerl GM, Vernooyl JHJ, van Spijki MP, van Suylen RJ, Pennings HJ, and Wouters EFM. Localization of granzyme A and B in lung specimensof patients with severe COPD. Eur Respir J 22: 45S, 2003.[CrossRef]
  41. Moss ML and Lambert MH. Shedding of membrane proteins by ADAM family proteases. Essays Biochem 38: 141-153, 2002.[ISI][Medline]
  42. Munch IC, Markussen NH, and Oritsland NA. Resting oxygen consumption in rats during food restriction, starvation, and refeeding. Acta Physiol Scand 148: 335-340, 1993.[ISI][Medline]
  43. Oved S and Yarden Y. Signal transduction: molecular ticket to enter cells. Nature 416: 133-136, 2002.[CrossRef][ISI][Medline]
  44. Pelkonen M, Notkola IL, Tukiainen H, Tervahauta M, Tuomilehto J, and Nissinen A. Smoking cessation, decline in pulmonary function and total mortality: a 30 year follow up study among the Finnish cohorts of the seven countries study. Thorax 56: 703-707, 2001.[Abstract/Free Full Text]
  45. Perrin BJ and Huttenlocher A. Calpain. Int J Biochem Cell Biol 34: 722-725, 2002.[CrossRef][ISI][Medline]
  46. Pistritto G, Jost M, Srinivasula SM, Baffa R, Poyet JL, Kari C, Lazebnik Y, Rodeck U, and Alnemri ES. Expression and transcriptional regulation of caspase-14 in simple and complex epithelia. Cell Death Differ 9: 995-1006, 2002.[CrossRef][ISI][Medline]
  47. Reddy P, Slack JL, Davis R, Cerretti DP, Kozlosky CJ, Blanton RA, Shows D, Peschon JJ, and Black RA. Functional analysis of the domain structure of tumor necrosis factor-{alpha} converting enzyme. J Biol Chem 275: 14608-14614, 2000.[Abstract/Free Full Text]
  48. Russell JH and Ley TJ. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 20: 323-370, 2002.[CrossRef][ISI][Medline]
  49. Ruvolo PP, Deng X, and May WS. Phosphorylation of Bcl 2 and regulation of apoptosis. Leukemia 15: 515-522, 2001.[CrossRef][ISI][Medline]
  50. Saetta M, Baraldo S, Corbino L, Turato G, Braccioni F, Rea F, Cavallesco G, Tropeano G, Mapp CE, Maestrelli P, Ciaccia A, and Fabbri LM. CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 160: 711-717, 1999.[Abstract/Free Full Text]
  51. Sahebjami H, Dynes R, and Massaro D. The effect of betamethasone on pressure-volume characteristics of nonfetal rat lungs. Am Rev Respir Dis 113: 493-496, 1976.[ISI][Medline]
  52. Sahebjami H and Wirman JA. Emphysema-like changes in lungs of starved rats. Am Rev Respir Dis 124: 619-624, 1981.[ISI][Medline]
  53. Scanlon PD, Connett JE, Waller LA, Altose MD, Bailey WC, and Buist AS. Smoking cessation and lung function in mild-to-moderate chronic obstructive pulmonary disease. The Lung Health Study. Am J Respir Crit Care Med 161: 381-390, 2000.[Abstract/Free Full Text]
  54. Scherle W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie 26: 57-60, 1970.[Medline]
  55. Seagrave J. Oxidative mechanisms in tobacco smoke-induced emphysema. J Toxicol Environ Health A 61: 69-78, 2000.[CrossRef][ISI][Medline]
  56. Sherrill DL, Enright PL, Kaltenborn WT, and Lebowitz MD. Predictors of longitudinal changes in diffusing capacity over 8 years. Am J Respir Crit Care Med 160: 1883-1887, 1999.[Abstract/Free Full Text]
  57. Silverman EK and Speizer FE. Risk factors for the development of chronic obstructive pulmonary disease. Med Clin North Am 80: 501-522, 1996.[ISI][Medline]
  58. Smith KM, Gaultier A, Cousin H, Alfandari D, White JM, and DeSimone DW. The cysteine-rich domain regulates ADAM protease function in vivo. J Cell Biol 159: 893-902, 2002.[Abstract/Free Full Text]
  59. StatMost Statistical Analysis and Graphics, Data Most Corporation.
  60. Stupina AS and Chernyi IaM. Stereologic analysis of the respiratory zone of the lungs of the laboratory rat and man during aging. Arkh Anat Gistol Embriol 88: 61-64, 1985.
  61. Subcommittee on Laboratory Animals Nutrition. In: Nutrient Requirements of Laboratory Animals (3rd ed.). Washington, DC: National Research Council, National Academy of Sciences, 1978, p. 47.
  62. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, and Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397: 441-446, 1999.[CrossRef][ISI][Medline]
  63. Tetley TD. Macrophages and the pathogenesis of COPD. Chest 121: 156S-159S, 2002.[Abstract/Free Full Text]
  64. Thet LA, Delaney MD, Gregorio CA, and Massaro D. Protein metabolism by rat lung: influence of fasting, glucose, and insulin. J Appl Physiol 43: 463-467, 1977.[Abstract/Free Full Text]
  65. Thurlbeck WM. The internal surface area of nonemphysematous lungs. Am Rev Respir Dis 95: 765-773, 1967.[ISI][Medline]
  66. Tremblay GM, Wolbink AM, Cormier Y, and Hack CE. Granzyme activity in the inflamed lung is not controlled by endogenous serine proteinase inhibitors. J Immunol 165: 3966-3969, 2000.[Abstract/Free Full Text]
  67. Viegi G, Sherrill DL, Carrozzi L, Di Pede F, Baldacc S, Pistelli F, and Enright P. An 8-year follow-up of carbon monoxide diffusing capacity in a general population sample of northern Italy. Chest 120: 74-80, 2001.[Abstract/Free Full Text]
  68. Vlahovic G, Russell ML, Mercer RR, and Crapo JD. Cellular and connective tissue changes in alveolar septal walls in emphysema. Am J Respir Crit Care Med 160: 2086-2092, 1999.[Abstract/Free Full Text]
  69. Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, Oezatalay D, and Uhal BD. Apoptosis of lung epithelial cells in response to TNF-{alpha} requires angiotensin II generation de novo. J Cell Physiol 185: 253-259, 2000.[CrossRef][ISI][Medline]
  70. Weibel ER. Stereological Methods. New York: Academic, 1979, p. 9-196.
  71. Wiebe BM and Laursen H. Lung morphometry by unbiased methods in emphysema: bronchial and blood vessel volume, alveolar surface area and capillary length. APMIS 106: 651-656, 1998.[ISI][Medline]
  72. Wikelski M and Thom C. Marine iguanas shrink to survive El Nino. Nature 403: 37-38, 2000.[CrossRef][ISI][Medline]
  73. Winick M. Hunger disease. In: Studies by the Jewish Physicians in the Warsaw Ghetto. New York: Wiley, 1979.
  74. Zhang W, Hoffman B, and Lieberman DA. Ectopic expression of MyD118/Gadd45/CR6 (Gadd45{beta}/{alpha}/{gamma}) sensitizes neoplastic cells to genotoxic stress-induced apoptosis. Int J Oncol 18: 749-757, 2001.[ISI][Medline]