We previously reported that genetically obese mice exhibit innate airway hyperresponsiveness (AHR) and enhanced ozone (O3)-induced pulmonary inflammation. Such genetic deficiencies in mice are rare in humans, and they may not be representative of human obesity. Thus the purpose of this study was to determine the pulmonary phenotype of mice with diet-induced obesity (DIO), which more closely mimics the cause of human obesity. Therefore, wild-type C57BL/6 mice were reared from the time of weaning until at least 30 wk of age on diets in which either 10 or 60% of the calories are derived from fat in the form of lard. Body mass was ∼40% greater in mice fed 60 vs. 10% fat diets. Baseline airway responsiveness to intravenous methacholine, measured by forced oscillation, was greater in mice fed 60 vs. 10% fat diets. We also examined lung permeability and inflammation after exposure to room air or O3 (2 parts/million for 3 h), an asthma trigger. Four hours after the exposure ended, O3-induced increases in bronchoalveolar lavage fluid protein, interleukin-6, KC, macrophage inflammatory protein-2, interferon-γ-inducible protein-10, and eotaxin were greater in mice fed 60 vs. 10% fat diets. Innate AHR and augmented responses to O3 were not observed in mice raised from weaning until 20–22 wk of age on a 60% fat diet. These results indicate that mice with DIO exhibit innate AHR and enhanced O3-induced pulmonary inflammation, similar to genetically obese mice. However, mice with DIO must remain obese for an extended period of time before this pulmonary phenotype is observed.
- bronchoalveolar lavage fluid
- lung elastance
obesity is an important public health problem that is associated with several respiratory diseases, including obesity-hypoventilation syndrome, obstructive sleep apnea, and asthma (14, 15, 53). Epidemiological studies indicate an increased incidence of asthma, wheezing, or airway hyperresponsiveness (AHR) in overweight or obese children, adolescents, and adults (14, 53). The relationship between obesity and asthma is likely to be a causal one, because longitudinal studies controlling for a number of potential confounders, including physical activity, indicate that the relative risk of incident asthma progressively increases with increasing body mass index and that obesity antedates asthma (6, 7, 18, 41). Furthermore, morbidly obese asthmatic individuals examined after diet- or surgically induced weight loss report a decrease in both the severity and symptoms of asthma (38, 42, 56, 57).
Our laboratory has been utilizing murine models of obesity to explore the mechanistic basis for the relationship between obesity and asthma. Our laboratory has reported that obese mice exhibit innate AHR (29, 30, 37, 48, 54). Obese mice also exhibit enhanced ozone (O3)-induced pulmonary inflammation (29, 37, 54). O3 is a common environmental pollutant that exacerbates asthma (13, 17). Innate AHR and enhanced pulmonary responses to O3 were observed in 1) mice obese as a result of a genetic deficiency in leptin, a satiety hormone (ob/ob mice) (30, 48, 54), 2) mice obese because of a genetic deficiency in the long isoform of the leptin receptor (db/db mice) (30, 37), and 3) mice obese due to a genetic deficiency in carboxypeptidase E (Cpe) (29), an enzyme involved in processing prohormones and proneuropeptides involved in satiety and energy expenditure (Cpefat mice) (33). Although each of these genetically obese mice manifests excessive adiposity, these genetic deficiencies are rare in humans (8, 12). Ob/ob, db/db, and Cpefat mice also develop very marked and rapid weight gain. For example, ob/ob and db/db mice have increased fat mass at 2 wk of age while Cpefat mice develop obesity by 8–12 wk of age (33). At 8–12 wk of age, ob/ob and db/db mice weigh nearly 150% more than lean, wild-type C57BL/6 controls (37, 54), whereas Cpefat mice weigh 90% more than C57BL/6 controls at 14–16 wk of age (29). Therefore, the purpose of this study was to determine whether innate AHR and increased pulmonary responses to O3 are also observed in mice in which obesity is induced through the consumption of a high-fat diet, a situation that more closely mimics the development of human obesity. Previous data demonstrate that feeding C57BL/6 mice a diet consisting of 60 kcal percent fat slowly leads to significant weight gain over several months compared with controls fed a diet consisting of 10 kcal percent fat (69). In addition, the more modest and less rapid increases in body weight observed in C57BL/6 mice follows a time course that better resembles the development of obesity in humans. Finally, feeding mice a diet consisting of nearly 50% fat has been shown to alter pulmonary responses to allergen (40), another important asthma trigger.
Accordingly, C57BL/6 mice were reared from the time of weaning on diets in which either 10 or 60% of the calories are derived from fat in the form of lard. C57BL/6 mice were used to allow for comparison with ob/ob, db/db, and Cpefat mice, which were on this background (29, 37, 54). Also, compared with other mouse strains, C57BL/6 mice are very susceptible to the development of diet-induced obesity (DIO) and obesity-related pathologies, including hyperglycemia, insulin resistance, elevated plasma cholesterol, and nonalcoholic steatohepatitis (23, 24, 34, 43, 52, 58). Measurements of pulmonary mechanics and airway responsiveness to intravenous (iv) methacholine were made at various time points during the induction of obesity. To examine the effect of DIO on inflammatory responses to O3, we also exposed lean and obese mice to O3 [2 parts/million (ppm) for 3 h], and measured protein, cytokines, chemokines, and cells in the bronchoalveolar lavage fluid (BALF) 4 h after the cessation of exposure.
Some of the results of this study have been previously reported in the form of an abstract (26).
MATERIALS AND METHODS
Fourteen-day-old male and female C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed with their mother in a rodent barrier facility until 21–28 days of age. At that time, the mice were weaned and divided into two groups. One group received a diet in which 10% of the calories are derived from fat in the form of lard, while the other group was fed a diet in which 60% of the calories are derived from fat in the form of lard (Research Diets, New Brunswick, NJ). Before and after weaning, the mice were housed in micro-isolator cages, where they were given food and water ad libitum and exposed to a 12:12-h light-dark cycle. Mice were studied when they were at least 20 wk of age. The Harvard Medical Area Standing Committee on Animals approved all of the experimental protocols used in this study.
Two experimental protocols were used in this study. In the first series of experiments, the effect of DIO on innate airway responsiveness to intravenous methacholine was assessed. For these experiments, mice were anesthetized and instrumented for the measurement of pulmonary mechanics as described below. In the second series of experiments, the effect of DIO on lung permeability and inflammation in response to acute O3 exposure was examined. For these experiments, mice were exposed to either room air or O3 (2 ppm) for 3 h. Four hours following the cessation of exposure, mice were euthanized, blood was collected via cardiac puncture, and bronchoalveolar lavage (BAL) was performed. Mice were examined either at 20–22 wk of age or when they were at least 30 wk of age. The O3 exposure protocol was chosen to allow for comparison with data of other investigators studying acute O3-induced pulmonary inflammation in mice (9, 10, 21, 25, 44, 51, 60, 72), including obese mice (29, 37, 54). We chose to examine the levels of inflammatory mediators in the BALF at 4 h following the cessation of O3 exposure because previous data from our laboratory indicated that the levels of many of these mediators are highest at this time in both wild-type and obese mice (29, 37, 54).
For acute (3 h) exposure to 2 ppm O3, conscious mice were placed into individual wire-mesh cages, which were subsequently placed inside a 145-liter stainless steel and Plexiglas exposure chamber. Exposure to room air was identical to the corresponding O3 exposure except that a separate and identical exposure chamber was used. The O3 was generated and the concentration monitored as previously described (27).
Measurement of pulmonary mechanics and airway responsiveness.
Mice were anesthetized with xylazine (7 mg/kg) and pentobarbital sodium (50 mg/kg). The trachea was cannulated with a tubing adaptor, and the tail vein was cannulated for the delivery of acetyl-β-methylcholine chloride (methacholine) (Sigma-Aldrich, St. Louis, MO). A wide incision in the chest wall was made bilaterally to expose the lungs to atmospheric pressure, and the mice were artificially ventilated at 150 breaths/min with a tidal volume (Vt) of 0.3 ml and a positive end-expiratory pressure (PEEP) of 3 cmH2O using a mouse ventilator (flexiVent, SCIREQ, Montreal, Quebec, Canada).
In some mice, quasi-static pressure-volume relationships were obtained using the flexiVent system to determine quasi-static lung elastance (E). The lungs were first inflated to three times Vt to standardize volume history. Progressive increments in volume, ∼0.11 ml, were introduced from end-expiratory volume using the flexiVent. Airway opening pressure was measured after each increment in volume was held for 1 s.
Measurements of baseline pulmonary mechanics and responses to iv methacholine were then obtained using the forced oscillation technique (flexiVent; SCIREQ), as described previously (29, 37, 48, 54). Mice were given a brief inflation to three times Vt to standardize volume history. One minute later, phosphate-buffered saline (PBS) was administered (1 μl/g), and total pulmonary resistance (Rl) was measured using a 2.5-Hz sinusoidal forcing function every eighth breath for the next 1–2 min, until Rl peaked. Because both the airways and the lung tissues can contribute to Rl, we also measured airway resistance (Raw) and the coefficients of lung tissue damping (G) and lung tissue elastance (H) in these mice. To do so, we used an 8-s optimized pseudorandom forcing function containing frequencies ranging from 0.25 to 19.63 Hz to obtain measurements of total lung impedance (Zl). A parameter estimation model (19) was used to partition Zl into components representing Raw, G, and H. Use of this model in mice has been verified by our laboratory and others (20, 46, 48, 64). Once measurements of the response to PBS were complete, the mouse was given another inflation to three times Vt, and the procedure was repeated using doses of methacholine dissolved in PBS increasing in approximate half-log intervals from 0.03 to 1.0 mg/ml at a dose of 1 μl/g. In some of the mice, we were unable to cannulate their tail veins for the delivery of methacholine. However, baseline pulmonary mechanics and quasi-static pressure volume curves were measured in all animals.
Blood was collected from the heart via cardiac puncture after euthanasia with pentobarbital sodium. The total number of blood leukocytes was determined as described previously (27). Serum was isolated and stored at −20°C and subsequently analyzed for adiponectin, eotaxin, leptin, and soluble tumor necrosis factor receptors (sTNFR) 1 and 2. The serum concentrations of these substances were determined with either ELISAs or DuoSet ELISA development kits (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions.
Immediately before BAL, the animal was euthanized with an overdose of pentobarbital sodium. For BAL, the trachea was exposed in situ, a small incision was made in the trachea, and a plastic catheter attached to a syringe was inserted. The lungs were lavaged twice with 1 ml of ice-cold lavage buffer, PBS containing 0.6 mM EDTA. The resulting lavagates were pooled and stored on ice until further use. The lavagate was spun, the supernatant was collected and stored at −80°C until further use, and the remaining cell pellet was resuspended in 1 ml of Hanks' balanced salt solution (Sigma-Aldrich). The total number of BALF cells was determined by counting the number of cells with a hemocytometer. Next, BALF cells were spun onto glass microscope slides using a Cytospin 3 Cytocentrifuge (Thermo Shandon, Pittsburgh, PA). Afterward, the slides were air-dried and stained with Hema 3 (Biochemical Sciences, Swedesboro, NJ), and at least 300 cells were counted under a light microscope for differential cell analysis.
Protein and enzyme-linked immunosorbent assays.
The total BALF protein concentration was determined spectrophotometrically according to the Bradford protein assay procedure (Bio-Rad Laboratories, Hercules, CA). The concentration of BALF eotaxin, interferon-γ-inducible protein (IP)-10, interleukin (IL)-6, KC, macrophage inflammatory protein-2 (MIP-2), sTNFR1, and sTNFR2 were determined with either enzyme-linked immunosorbent assays (ELISAs) or DuoSet ELISA development kits (R&D Systems), according to the manufacturer's instructions.
Comparisons of pulmonary mechanics, quasi-static elastance, and serum markers of inflammation were made by unpaired Student's t-tests. Comparisons of BALF parameters were assessed using factorial ANOVA, using diet and exposure as the main effects. Fisher's least significant difference test was used as a follow-up to determine the significance of differences between individual groups. STATISTICA software (StatSoft, Tulsa, OK) was used to perform all statistical analyses. The results are expressed as means ± SE, except where noted. A P value <0.05 was considered significant.
Effect of DIO on body mass.
Mice fed a diet consisting of 60% fat from weaning weighed significantly more than mice fed a diet consisting of 10% fat (Table 1). At 20–22 wk of age, mice fed a 60% fat diet weighed ∼37% more than mice fed a 10% fat diet, while mice 30–38 wk of age weighed ∼39% more than mice fed a 10% fat diet.
Effect of DIO on pulmonary mechanics and airway responsiveness.
At 20–22 wk of age, there was no effect of diet on baseline airway and parenchymal oscillation mechanics (Rl, Raw, G, and H) (Table 1). In mice that were at least 30 wk of age and older, baseline Rl, Raw, and H were greater in mice reared on a 60 vs. a 10% fat diet.
In 20- to 22-wk-old mice, there was no difference in airway responsiveness to methacholine between mice fed 10 vs. 60% fat diets (Fig. 1A). In contrast, in mice at least 30 wk of age and older, there was a significant effect of diet on airway responsiveness to methacholine: for all concentrations of methacholine, with the exception of 1 mg/ml, mice fed a 60% fat diet had significantly greater Rl responses (Fig. 1B) than mice fed a 10% fat diet. To determine the locus (airways vs. parenchyma) of this diet-induced change in responsiveness, we also measured methacholine-induced changes in Raw, G, and H (Fig. 2). Methacholine administration increased Raw in both diet groups. However, the increases in Raw were significantly greater in the mice fed a 60 vs. 10% fat diet (Fig. 2A). There were no methacholine-induced changes in G or H in mice fed either diet (Fig. 2, B and C).
Effect of DIO on quasi-static pressure-volume curves.
Our laboratory has previously reported that the quasi-static lung elastance (E) of genetically obese mice is increased compared with lean, wild-type controls (29, 37). To determine whether dietary obesity impacted E, we generated quasi-static pressure-volume (PV) curves in open-chested, wild-type (C57BL/6) mice fed a diet consisting of either 10 or 60% fat (Fig. 3, A and B). There was no effect of diet on quasi-static lung elastance in mice 20–22 wk of age or in mice 30 wk of age and older (Table 1).
Effect of DIO on O3-induced lung hyperpermeability and inflammation.
Following exposure to room air, there was no effect of DIO on any of the outcome indicators analyzed in the BALF in mice 30 wk of age and older (Figs. 4 and 5). O3 causes disruption of the lung epithelial barrier leading to increased permeability that is routinely assessed by determining the levels of BALF protein (2, 3, 65). To determine the effect of DIO on O3-induced lung hyperpermeability, we examined the levels of BALF protein 4 h following the cessation of O3 exposure. O3 exposure increased the levels of BALF protein (Fig. 4A). However, the levels of BALF protein were increased to a greater extent in mice with DIO. We also examined the effect of DIO on O3-induced pulmonary inflammation by determining the number of BALF neutrophils, epithelial cells, and macrophages following the cessation of O3 exposure (Fig. 4, B, C, and D). In both dietary groups, O3 exposure increased the number of BALF neutrophils and epithelial cells, but decreased the number of BALF macrophages. However, there was no effect of diet on any of these outcome indicators.
Our laboratory has previously reported that IL-6 deficiency ameliorates O3-induced increases in BALF protein (28). Thus we investigated whether the elevated levels of BALF protein were associated with increased levels of IL-6. Compared with air, O3 exposure increased the levels of BALF IL-6 (Fig. 5); however, the effect was significantly greater in the 60 vs. the 10% fat-fed mice. Because we have observed greater increases in chemokines in obese ob/ob, db/db, and Cpefat vs. lean, wild-type mice exposed to O3 (29, 37, 54), we also examined the effect of dietary obesity on BALF eotaxin, KC, MIP-2, and IP-10 (Fig. 5). Four hours following O3 exposure, the levels of BALF eotaxin, KC, MIP-2, and IP-10 were significantly greater in mice fed a 60 vs. 10% fat diet. Finally, because IL-6 can influence the levels of sTNFR1 and sTNFR2 (28, 63) and because IL-6 expression is increased in mice with DIO, we determined the BALF levels of these sTNFRs in mice fed diets consisting of either 10 or 60% fat. Following exposure to O3, the levels of BALF sTNFR1 and sTNFR2 were greater in mice fed a 60 vs. 10% fat diet.
In contrast to the mice 30 wk of age and older, there was no effect of diet on O3-induced changes in BALF protein, cells, cytokines or chemokines in mice 20–22 wk of age (data not shown).
Effect of DIO on systemic markers of inflammation.
Our laboratory and others have previously reported that markers of systemic inflammation, including certain proinflammatory cytokines and chemokines, soluble cytokine receptors, and total blood leukocytes, are elevated in the serum of genetically obese mice (22, 29, 30, 37). To determine whether DIO also leads to the development of systemic inflammation, we measured the serum levels of sTNFR1, sTNFR2, eotaxin, leptin, and adiponectin as well as the total number of blood leukocytes in air exposed, wild-type (C57BL/6) mice fed a diet consisting of either 10 or 60% fat (Fig. 6), which were 30 wk of age and older. DIO significantly increased the serum levels of sTNFR2 and leptin. However, the levels of sTNFR1 and eotaxin as well as the total number of blood leukocytes were unaffected by DIO. In addition, DIO had no effect on the levels of the anti-inflammatory hormone, adiponectin.
These results indicate that diet-induced obesity leads to the development of innate AHR (Fig. 1B), and to enhanced O3-induced lung permeability and inflammation (Figs. 4 and 5). These data are consistent with our laboratory's previous observations in genetically obese ob/ob (30, 48, 54), db/db (30, 37), and Cpefat mice (29). However, our data indicate that C57BL/6 mice must consume a high-fat diet and remain obese for an extended period of time before the pulmonary phenotype characteristic of obese mice is observed.
In this study, C57BL/6 mice fed a diet consisting of 60% fat from weaning until at least 20 wk of age weighed nearly 40% more than age- and sex-matched mice fed a 10% fat diet for the same time period. The increase in body mass observed in C57BL/6 mice fed a high-fat diet was more modest and less rapid than those exhibited by genetically obese ob/ob, db/db, and Cpefat mice (29, 30, 37, 54). The time period required for DIO to develop and the magnitude of the increase in body mass we observed in C57BL/6 mice with DIO is consistent with other studies (4, 24, 69). Similar to genetically obese mice, the increase in the total body mass of C57BL/6 mice with DIO is almost entirely due to an increase in fat mass (4, 5). Furthermore, C57BL/6 mice with DIO manifest several obesity-related pathologies, including hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, elevated plasma cholesterol, and nonalcoholic steatohepatitis (23, 24, 34, 35, 43, 52, 58). Of these obesity-related pathologies, we only examined the levels of serum leptin, and as expected, C57BL/6 mice with DIO had increased levels of serum leptin (Fig. 6).
Mice with DIO, which were maintained on a 60% fat diet from weaning until at least 30 wk of age, had increased airway responsiveness to methacholine compared with age- and sex-matched mice maintained on a 10% fat diet. Innate AHR to nonspecific bronchoconstrictors is also a consistent feature of genetically obese mice (29, 30, 37, 48, 54). The locus of the innate AHR observed in mice with DIO was the airways, rather than the lung parenchyma, because G and H, parameters reflecting the mechanics of the lung tissue, did not change with methacholine administration (Fig. 2). The airways are also the locus of the innate AHR observed in ob/ob and Cpefat mice (29, 48).
Breathing at low lung volumes has been shown to enhance airway responsiveness to methacholine in human subjects (11), and because of alterations in the chest wall, functional residual capacity is reduced in obese compared with lean human subjects (70, 71). Obese subjects also breathe with lower Vt than lean subjects (49) and changes in Vt can also affect airway responsiveness (11). However, it is unlikely that differences in either absolute or tidal lung volumes can explain the innate AHR observed in mice with DIO: we removed the impact of the chest wall by opening the chest wall and applying a fixed PEEP, and all mice were artificially ventilated with the same Vt. Although obesity can also reduce absolute lung size in mice (37), the observation that lung PV curves were not affected by DIO (Fig. 3) suggests that lung size was not affected by DIO. In contrast to the lack of effect of DIO on E (Fig. 3, Table 1), our laboratory has previously reported that E is increased in db/db and Cpefat mice compared with lean controls (29, 37). Db/db and Cpefat mice weigh ∼150 and 90% more, respectively, than age- and sex-matched wild-type controls, whereas we observed only an approximate 40% increase in body weight in these mice with DIO (Table 1). Hence, it is possible that obesity does not impact E until a more substantial amount of adipose tissue has accumulated. In support of this notion, there is an inverse relationship between body mass index and lung volumes in human subjects (71). Interestingly, in mice 30 wk of age and older, DIO did increase H (Table 1) even though it did not significantly alter E (Fig. 3, Table. 1). There was also a small but significant increase in Raw in these mice (Table 1). If this airway narrowing was not uniformly distributed in the lung, it would result in lung units with longer time constants (higher resistances) effectively dropping out during oscillation at high frequencies, an event that would increase H even though the “true” elastic properties of the lung tissue were not altered.
As previously discussed, it is possible that the innate AHR observed in mice with DIO may be related to chronic, low-grade systemic inflammation. In obese humans, there are increased serum concentrations of cytokines, cytokine receptors, chemokines, and acute-phase proteins that correlate with the presence of diseases common to obesity, including Type 2 diabetes and atherosclerosis (1, 45, 61, 67). Our results (Fig. 6) also indicate increased serum concentrations of leptin and sTNFR2 in mice fed a diet consisting of 60 vs. 10% fat. Similarly, increases in some markers of inflammation are observed in the serum of other types of obese mice (29, 30, 37). Increased serum leptin cannot explain the development of innate AHR in all types of obese mice. For example, ob/ob mice exhibit innate AHR, but they are leptin deficient (30, 48, 54). However, it is still conceivable that leptin may be important for AHR in mice with DIO. Exogenous leptin has been shown to augment the AHR induced by allergen challenge in mice (55). In addition, leptin has the capacity to either induce or augment TNF-α expression (16, 36, 50). Exogenous TNF-α can induce AHR (31, 62). Thus the increased levels of leptin may enhance TNF-α expression and lead to the development of AHR. Indeed, Lang and colleagues demonstrate that neutralization of TNF-α with an anti-TNF-α antibody attenuates the innate AHR of obese db/db and Cpefat mice (32). On the other hand, mice with DIO examined at either 20–22 wk of age did not exhibit innate AHR. These mice had approximately the same percent increase in body mass as the mice 30 wk of age and older with DIO and also had increases in serum leptin (102.4 ± 9.4 vs. 30.6 ± 9.9 ng/ml in mice fed 60 vs. 10% fat diets; P < 0.001). Others have reported that the adipose tissue of even 16-wk-old mice with DIO is infiltrated with macrophages and exhibits heightened expression of proinflammatory cytokines and chemokines (69). Therefore, innate AHR does not coincide temporally with the onset of chronic systemic inflammation. Thus, it appears that if the low-grade, systemic inflammation of obesity accounts in any way for the pulmonary phenotype of mice with DIO, it must be present for an extended period of time before this phenotype is manifest.
Consistent with other types of obese mice (29, 37, 54), mice with DIO exhibit enhanced O3-induced lung permeability (Fig. 4A), as well as enhanced O3-induced pulmonary expression of IL-6, eotaxin, KC, MIP-2, and IP-10 (Fig. 5). Despite the increased BALF levels of KC, MIP-2, and IP-10, chemokines known to be important for recruitment of neutrophils following acute O3 exposure (27, 39), there were no differences between the dietary groups in the emigration of neutrophils to the airspaces (Fig. 4B). Similarly, we observed no difference in BALF neutrophils between wild-type and ob/ob mice following O3 exposure, whereas the O3-induced influx of neutrophils is increased in db/db and Cpefat vs. wild-type mice (29, 37). We do not know what accounts for the inability of the increased BALF chemokines to recruit more neutrophils in ob/ob mice and in these DIO mice, but it is possible that there are alterations in neutrophil adhesion molecules or heightened levels of neutrophil chemokines in the blood that impact neutrophil migration into the lungs and airways.
It is unlikely that differences in the inhaled dose of O3 account for the enhanced effects of O3 on BALF protein, cytokines, and chemokines in mice with DIO. The inhaled dose of O3 is the product of O3 concentration, exposure duration, and minute ventilation (68), and our laboratory has previously reported no difference in minute ventilation in mice with DIO (53). It is possible that the augmented effects of O3 on cytokine and chemokine expression in mice with DIO are the result of differences in lipids in the epithelial cell membranes or in the lung surface liquid, leading to greater or altered lipid peroxidation, the initial step in the inflammatory cascade brought about by O3 (47).
As was the case for AHR, the augmented responses to O3 were observed in mice with DIO that were studied at 30 wk of age and older, but not in mice studied at 20–22 wk of age, even though both groups of mice had approximately the same increase in body mass. These data suggest that in this model, augmented responses to O3 are not dependent on the amount of weight gained, but rather, on the amount of time that the animal remains obese. From our data, we cannot determine the reason why the length of time obesity persists is important for the development of innate AHR or augmented responses to O3.
Although mice with DIO manifest many aspects of the pulmonary phenotype characteristic of genetically obese mice, this model was not ideal. First, this model does not allow us to separate the effects of an increased intake of lipids vs. an increased body mass on the observed responses in mice with DIO. However, our observations in mice with DIO are qualitatively similar to genetically obese mice, which were fed a diet with a much lower fat content than 60%, suggesting that the obesity rather than the high lipid content of the diet contributed to the observed responses. Second, because of the extended period of time required to induce pulmonary changes in this model, and the sedentary life of these mice, even the 10% fat-fed mice were quite heavy. These mice weighed as much as 15 g more than the much younger C57BL/6 controls used in our previous studies of genetically obese mice (29, 37). In addition, in these 10% fat-fed mice, serum leptin, a marker of adiposity, averaged nearly 25 ng/ml (Fig. 6D), compared with 5–10 ng/ml in our laboratory's previous studies of younger C57BL/6 mice (29, 37). Essentially, while we were able to observe differences between the 60 and 10% fat fed mice, it is possible that these actually represent differences between obesity (60% fat) and overweight (10% fat). This may explain why we were unable to observe differences in serum adiponectin (Fig. 6). The DIO model appears to be quite suitable for studying the metabolic effects of obesity (58, 59, 66, 69), but the pulmonary phenotype may be less sensitive and require either more substantial obesity (such as we observe in genetically obese mice) or less marked obesity but of longer duration. Nevertheless, the value of this model is that it permits the generation of obesity in mice with other genetic deficiencies that may be useful for understanding the mechanistic basis for the relationship between obesity and asthma.
In conclusion, we report that mice with DIO exhibit innate AHR and enhanced O3-induced lung permeability and inflammation compared with 10% fat-fed controls. These effects appear to be dependent on the consumption of a high-fat diet for an extended period of time and/or obesity of fairly long duration. Taken together with our results from genetically obese mice, these data suggest that innate AHR and enhanced O3-induced pulmonary responses are consistent features of obese mice and not dependent on the modality of obesity induction.
This study was supported by National Heart, Lung, and Blood Institute Grant HL-084044 (to S. A. Shore), National Institute of Environmental Health Sciences Grants ES-013307 (to S. A. Shore) and ES-00002 (to J. D. Brain), American Lung Association Research Training Fellowship RT-41-N (to R. A. Johnston), and a generous gift from Paul and Mary Finnegan.
The authors thank Dr. James P. Butler for discussions on the impact of obesity on pulmonary mechanics.
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.
- Copyright © 2008 the American Physiological Society