MCHeC/eC Mice Are Resistant to Aging-Associated Increases in Body Weight and Insulin Resistance
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糖尿病学杂志 2006年第2期
1 Division of Endocrinology, Department of Medicine, Beth Israel Medical Center, Boston, Massachusetts
2 Research Division, Joslin Diabetes Center, Boston, Massachusetts
3 Department of Medicine, Harvard Medical School, Boston, Massachusetts
4 Department of Sport and Leisure Studies, Yonsei University, Seoul, Korea
5 Division of Gastroenterology, Department of Medicine, Beth Israel Medical Center, Boston, Massachusetts
Key Words: AUC, area under the curve DEXA, dual-energy X-ray absorptiometry GTT, glucose tolerance test ITT, insulin tolerance test MCH, melanin-concentrating hormone REE, resting energy expenditure Sir2, silent inflammatory regulator 2
ABSTRACT
Ablation of the hypothalamic peptide, melanin-concentrating hormone (MCH), leads to a lean phenotype and resistance to diet-induced obesity. Observation of MCHeC/eC mice at older ages suggested that these effects persist in mice >1 year old. Leanness secondary to caloric restriction is known to be associated with improved glucose tolerance as well as an overall increase in life span. Because the MCHeC/eC model represents leanness secondary to increased energy expenditure rather than caloric restriction, we were interested in determining whether this model of leanness would be associated with beneficial metabolic effects at older ages. To assess the effects of MCH ablation over a more prolonged period, we monitored male and female MCHeC/eC mice up to 19 months. The lean phenotype of MCHeC/eC mice persisted over the duration of the study. At 19 months, MCHeC/eC male and female mice weighed 23.4 and 30.8% less than their wild-type counterparts, a result of reduced fat mass in MCHeC/eC mice. Aged MCHeC/eC mice exhibited better glucose tolerance and were more insulin sensitive compared with wild-type controls. Aging-associated decreases in locomotor activity were also attenuated in MCHeC/eC mice. We also evaluated two molecules implicated in the pathophysiology of aging, p53 and silent inflammatory regulator 2 (Sir2). We found that expression of the tumor suppressor protein p53 was higher in MCHeC/eC mice at 9 and 19 months of age. In contrast, expression of Sir2 was unchanged. In aggregate, these findings suggest that MCH ablation improves the long-term outcome for several indicators of the aging process.
Advancing age is associated with increased susceptibility to numerous diseases. A progressive increase in visceral adiposity is a common feature of aging, and epidemiological evidence supports a role of adiposity as a prominent risk factor for metabolic disorders such as insulin resistance, type 2 diabetes, and cardiovascular disease (1eC3). Reversal of obesity through caloric restriction, lifestyle modifications, drug therapy, and surgical removal of visceral fat has beneficial effects on insulin resistance and other metabolic disorders associated with aging (4,5). Similarly, in rodent models, targeted deletion of specific genes has led to reductions in mouse body weights with concomitant improvements in their overall metabolic profiles (6eC8). However, information on the long-term effects of gene deletioneCinduced weight loss on aging-associated metabolic diseases and on other indicators of the aging process is very limited.
Melanin-concentrating hormone (MCH), which is expressed in the lateral hypothalamus, has emerged as an important regulator of energy balance and is also implicated in other behaviors such as water ingestion and anxiety (9eC11). Chronic infusion of MCH leads to excess weight gain and a shift of the animal to a "lipogenic" state associated with decreased energy expenditure and increased energy storage (12,13). When MCH was overexpressed in the lateral hypothalamus of mice, both an increase in food intake and weight gain were observed (14). On the other hand, ablation of both the MCH gene (MCHeC/eC) and the rodent receptor for MCH, designated MCHR-1, leads to a lean phenotype as a consequence of increased locomotor activity and increased basal metabolic rate (6,15). When MCH was genetically ablated, two other neuropeptides, which are derivatives of prepro-MCH and are designated N-EI and N-GE, were also ablated. Neither of these peptides has an effect on feeding alone, in combination with each other, or in combination with MCH (E.M.-F., unpublished data). Although a potential role of these peptides in the lean phenotype of the MCHeC/eC mouse has not been fully excluded, such a role is unlikely because the phenotype of mice lacking the MCH receptor is very similar to that of mice lacking the ligand (15).
Although we have found that MCHeC/eC mice are lean, hyperactive, and hypermetabolic (6,16,17), it is unknown whether this phenotype is maintained as the animals age. Leanness secondary to caloric restriction is known to be associated with improved glucose tolerance as well as with an increased life span. Because MCHeC/eC mice are lean secondary to increased energy expenditure rather than caloric restriction, we sought to determine whether MCH ablation would result in improved long-term outcomes for adiposity and insulin sensitivity, as well as for other indicators of the aging process. Therefore, we evaluated the phenotype of MCHeC/eC mice up to 19 months old. Parameters assessed included food intake, body weight, locomotor activity, and insulin sensitivity. In addition, we initiated an evaluation of potential long-term effects of MCH ablation on two key molecular markers linked to cellular senescence and organismal aging, namely the tumor suppressor protein p53 and the silent inflammatory regulator 2 (Sir2) protein, a member of the conserved sirtuin family of nicotinamide adenine dinucleotideeCdependent deacetylases (18,19). p53 is an important regulator of cell growth, apoptosis, and DNA repair (20,21), and numerous lines of evidence indicate that p53 plays divergent roles in the aging process (22eC24). It has also been reported that p53-deficient mice, which die at relatively young ages because of malignancies, show accelerated age-related accumulation of mutations in spleen and liver tissues (25). With regard to the role of Sir2 in aging, numerous studies in yeast and Drosophila have shown that caloric restriction increases longevity at least in part by increasing the activity and expression of Sir2 protein (26eC28). A recent report suggested that Sir2 homologs may also be important for longevity in humans (29). Furthermore, a human Sir2 homolog has been shown to bind to p53 and to downregulate p53-induced apoptosis (30,31). Therefore, we measured Sir2 and p53 protein levels in liver and spleen tissues from 9- and 19-month-old MCHeC/eC mice and their wild-type littermates, with the hypothesis that an attenuated aging phenotype in lean MCHeC/eC mice might be mediated in part via effects on these proteins.
RESEARCH DESIGN AND METHODS
These studies were approved by the Joslin Diabetes Center Animal Care and Use Committee. Mice lacking the gene for prepro-MCH were generated as previously described (6). For the current studies, MCHeC/eC mice were backcrossed onto a C57BL/6 background for eight generations. Mice were housed up to four per cage in the Joslin Animal Facility and maintained at 22°C, under an alternating 12-h light/dark cycle. Mice were placed on a normal diet (Purina Lab Diet 5008; 3.5 kcal/g) and allowed access to food and water ad libitum. Animals were monitored over a period of 19 months.
Glucose tolerance tests.
Intraperitoneal glucose tolerance tests (GTTs) were performed at the ages of 10 and 16 months. Mice were fasted overnight (1700eC0800) and were subsequently injected with glucose (2 g/kg body wt i.p.). Tail blood was collected at eC1, 15, 30, 60, and 120 min. Blood glucose concentrations were measured using a glucometer (Elite; Bayer, Mishawaka, IN).
Insulin tolerance tests.
Insulin tolerance tests (ITTs) were performed at the ages of 10 and 16 months. Mice were fasted for 3 h and subsequently injected with regular insulin (1 unit/kg; Eli Lilly and Co., Indianapolis, IN). Tail-blood samples were collected just before insulin administration and at 15, 30, 60, and 120 min after the insulin injection.
Oxygen consumption.
Oxygen consumption of 6- and 18-month-old mice was measured in eight open-circuit Oxymax chambers that are a component of the Comprehensive Laboratory Animal Monitoring System (Columbus Instruments, Columbus, OH). Mice were housed individually and were maintained at 24°C under a 12-h light/dark cycle (light period 0800eC2000) with free access to food and water. All mice were acclimatized to monitoring cages for 24 h before beginning an additional 24 h of hourly recordings of physiological parameters.
Resting energy expenditure.
Resting energy expenditure (REE) was determined by defining periods of inactivity and measuring oxygen consumption during those periods. Intervals during which animals showed <50 beam breaks were considered inactive intervals.
Locomotor activity.
Ambulatory activity of individually housed mice was evaluated on a relative basis using an eight-cage rack OPTO-M3 Sensor system (Columbus Instruments). Consecutive photobeam breaks occurring in adjacent photobeams were scored as an ambulatory movement. Cumulative ambulatory activity counts were recorded every hour for 24 h.
Body composition.
Fat and lean body mass were assessed using dual-energy X-ray absorptiometry (DEXA; Lunar PIXImus2 mouse densitometer; GE Medical Systems, Madison, WI) as described by the manufacturer. Mice were anesthetized by intraperitoneal injection of a 1:1 mixture of tribromoethanol:tert-amyl alcohol (0.015 ml/g body wt) and scanned, and total body fat and lean body mass were determined using an analysis program provided by the manufacturer.
Immunoblotting.
Liver and spleen tissues were homogenized in lysis buffer (20 mmol/l Tris-Cl, pH 7.5, 150 mmol/l NaCl, 1 mmol/l EDTA, and 1% Triton X-100) supplemented with various protease inhibitors. Protein concentration of the extracts was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total protein (25 e蘥) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with a mouse monoclonal -p53 antibody (Abcam, Cambridge, MA) or with a rabbit polyclonal -Sir2 antibody (Upstate USA, Lake Placid, NY). Signals were visualized using the Super Signal West Pico chemiluminescence reagent (Pierce, Rockford, IL).
Statistics.
Values are reported as group means ± SE. Interactions between genotype and physiological parameters were analyzed by two-way ANOVA. One-way ANOVA and independent t test were also used where appropriate. A P value of <0.05 was considered statistically significant. Statistical comparisons were made using Statview software (Abacus Concepts, Berkley, CA).
RESULTS
MCHeC/eC mice have a lean phenotype because of increased physical activity and energy expenditure.
Growth curves showed that MCH-ablated mice had a marked attenuation of weight gain normally associated with aging in both males and females (Fig. 1A and B). At 19 months of age, male MCHeC/eC mice weighed 32.6 ± 1.5 g, and male wild-type mice weighed 42.9 ± 1.5 g. Similarly, female MCHeC/eC mice weighed 31.2 ± 3.1 g, and female wild-type mice weighed 45.1 ± 2.6 g (P < 0.05). Body fat measurement by DEXA analysis showed that most of the weight difference was due to reduced fat mass (Fig. 1C and D). Dissection of MCHeC/eC mice revealed a marked reduction in perigonadal white adipose tissue (Fig. 1E) (MCHeC/eC males, 807 ± 171; wild-type males, 2,943 ± 281; MCHeC/eC females, 1,381 ± 879 mg; and wild-type females, 6,039 ± 1,108 mg; P < 0.05). The weight of other major organs such as liver (Fig. 1F) was not affected after correcting for total body weight.
The attenuated body weight gain in MCHeC/eC mice was not due to reduced feeding, because there was no significant difference in total food intake (food intake at 18 months; Fig. 1G and H). Food intake was also measured at 2, 7, and 10 months, and no significant difference in food intake was seen at any of these time points (not shown). This finding confirms a previous report from our laboratory showing no difference in food intake in younger male MCHeC/eC and male wild-type mice on a pure C57BL/6 background (32). Leanness in MCHeC/eC mice was observed at both young and old ages and was secondary to increases in both locomotor activity and basal metabolic rate (Fig. 2A and B). Analysis of locomotor activity showed that older wild-type mice had significantly reduced locomotor activity compared with younger mice. However, MCHeC/eC mice maintained increased locomotor activity at older ages (Fig. 2C) and displayed a resting metabolic rate comparable with younger animals. In addition, REE was reduced with aging by 11% in wild-type mice, whereas older MCHeC/eC mice had REE levels comparable with those of younger MCHeC/eC mice (Fig. 2D).
MCHeC/eC mice are resistant to aging-associated glucose intolerance.
A series of GTTs and ITTs were performed on both male and female MCHeC/eC mice at different ages. There was no difference in glucose tolerance in either male or female MCHeC/eC mice compared with their wild-type counterparts when they were 2 months old. Improved glucose tolerance was observed in male MCHeC/eC mice at 10 months of age when these animals showed a 28% reduction in glucose area under the curve (AUC) during GTTs compared with wild-type littermates (Fig. 3A). This difference in glucose tolerance in males became even more evident as they continued to age. As shown in Fig. 3B, at 16 months of age, male wild-type mice had 41.7% higher glucose AUC compared with their MCHeC/eC counterparts. At 10 months, female MCHeC/eC mice had similar glucose tolerance as their wild-type counterparts (Fig. 3C). However, a marked difference in glucose tolerance also manifested itself in female MCHeC/eC mice compared with their wild-type counterparts at 16 months. As shown in Fig. 3D, at this age, female wild-type mice had 108% higher glucose AUC compared with female MCHeC/eC mice. Two-way ANOVA repeated measurements showed that both male and female wild-type mice had significantly worse glucose tolerance than MCHeC/eC mice (P = 0.01). In addition, ITTs revealed that wild-type male mice were more insulin resistant compared with their MCHeC/eC counterparts (Fig. 3E and F). Plasma insulin levels during GTTs were significantly lower in 16-month-old MCHeC/eC mice compared with wild-type controls (Fig. 3G). At 16 months of age, consistent with their better insulin sensitivity, MCHeC/eC mice had substantially lower fasting insulin levels compared with wild-type animals. This effect was seen in both male and female animals (Fig. 4A and B).
MCHeC/eC mice show attenuation of tissue-specific aging-associated decreases in the tumor suppressor protein p53.
To begin defining potential anti-aging effects of MCH ablation at a molecular level, we measured Sir2 and p53 levels in liver and spleen tissues by immunoblotting. No significant difference in Sir2 protein levels was seen at any age between wild-type and MCHeC/eC mice in either liver or spleen (not shown). In contrast, as shown in Fig. 5A and B, aging was associated with a decline in p53 levels that was seen in both 19-month-old wild-type and MCHeC/eC mice, compared with 9-month-old animals. This effect was noted in both liver (9-month-old wild type, 1 ± 0.38; 9-month-old MCHeC/eC, 1.58 ± 0.17; 19-month-old wild type, 0.38 ± 0.08; and 19-month-old MCHeC/eC, 1.28 ± 0.21; P < 0.05) and spleen (9-month-old wild type, 1 ± 0.04; 9-month-old MCHeC/eC, 2.23 ± 0.4; 19-month-old wild type, 0.5 ± 0.2; and 19-month-old MCHeC/eC, 1.30 ± 0.2; P < 0.05). However, the progressive decline in p53 levels was attenuated in MCHeC/eC mice compared with their wild-type counterparts.
DISCUSSION
We previously reported that MCHeC/eC mice on a mixed genetic background (C57BL/6 and 129) were lean and hypophagic (6). Here, we have studied mice backcrossed eight generations onto a C57BL/6 background and monitored up to 19 months of age. On this background, MCHeC/eC mice are lean, hyperactive, and insulin sensitive compared with their wild-type littermates up to 19 months. As previously reported in young C57BL/6 mice lacking MCH, the lean phenotype was not due to a reduction in food intake. Measurements of food intake showed that MCHeC/eC mice had similar food intake to their wild-type counterparts. Reductions in body weight in MCHeC/eC mice on a C57BL/6 background were secondary to increased energy expenditure, resulting from both increased locomotor activity and basal metabolic rate. In MCHeC/eC mice, both were sustained at higher levels at older ages, while progressively declining in wild-type mice; however, the neuroendocrine systems mediating these changes downstream of MCH neurons remain unknown.
The increase in resting metabolic rate in MCHeC/eC mice may be due to increased thermogenesis, which in turn may be a consequence of increased sympathetic nervous system activity. We have previously found higher uncoupling protein-1 levels in brown adipose tissue from MCHeC/eC mice compared with their wild-type counterparts (16). Furthermore, mice lacking MCHR-1 also show a hypermetabolic phenotype due to increased autonomic activity (15,33).
A progressive increase in visceral adiposity is a common feature of aging (1,34). Increased visceral fat contributes to increased insulin resistance and reversal of visceral obesity via caloric restriction and/or surgical removal of visceral fat increases insulin sensitivity (35,36). In the current study, we found that 19-month-old MCHeC/eC mice did not develop glucose intolerance and maintained glucose sensitivity comparable with young wild-type mice. Because the amount of fat in MCHeC/eC mice was markedly reduced in both males and females compared with wild-type mice, we hypothesize that the more sensitive glucose tolerance phenotype seen in MCHeC/eC mice may be due to reduced visceral fat mass. Increased visceral adiposity with aging has been observed in animals and in humans, and surgical removal of visceral fat in 20-month-old rats was sufficient to restore peripheral and hepatic insulin action to levels seen in young rats (5,34,35). Therefore, the lack of visceral adiposity in older MCHeC/eC mice may explain the preservation of glucose tolerance and insulin sensitivity seen in these animals.
In mammals, caloric restriction delays the onset of aging-associated disorders, including cancer, atherosclerosis, and diabetes and can greatly increase life span (37eC41). As described earlier, both Sir2 and p53 are implicated in aging at the cellular and organismal levels. Sir2 genes play a central role in evolutionarily conserved pathways of aging and longevity. In yeast and Caenorhabditis elegans, life span is extended by the presence of extra copies of the SIR2/Sir-2.1 genes (42). However, little is known regarding the role of Sir2 in mammalian aging. Therefore, we measured Sir2 protein levels in 9- and 19-month-old MCHeC/eC mice and their wild-type littermates. We evaluated liver and spleen tissues from these animals, as we were examining Sir2 protein levels in tandem with p53 protein levels in these tissues on the basis that Sir2 is a negative regulator of p53 activity and p53-null mice are reported to have accelerated age-related occurrences of mutations in these two organs (25). Furthermore, the liver represents a primary target organ for obesity- and insulin resistanceeCinduced metabolic and inflammatory changes. In contrast to a report of increased Sir2 protein expression in long-term calorie-restricted rats (18), there was no difference in Sir2 protein expression between MCHeC/eC mice and wild-type mice, suggesting that leanness in MCHeC/eC mice does not upregulate Sir2 protein expression.
Advancing age is also associated with an increased risk of cancer and cellular senescence in response to oncogenic signals (43). In humans, the incidence of cancer rises exponentially in the final decades of life, resulting in a cumulative life time risk of one in two for men and one in three for women (43). As previously discussed, the p53 gene encodes a potent tumor suppressor protein, which induces cell cycle arrest and apoptosis and is implicated as a mediator of organismal aging as well. Results to date indicate that p53 protein levels are differentially regulated in a tissue-specific pattern during aging. For example, some studies have shown increased p53 levels in the gastric mucosa of rats aged up to 24 months (44), whereas others have shown a decline in p53 levels in the colonic mucosa (45) or no change in myocyte p53 levels in rats aged up to 24 months (46). Interestingly, we observed in both wild-type and MCHeC/eC mice that p53 protein levels in spleen and liver were substantially decreased at 19 months compared with at 9 months. To our knowledge, this is the first report showing an aging-associated decrease in p53 protein levels in mouse liver and spleen. However, aged MCHeC/eC mice still retained higher p53 levels in both liver and spleen than wild-type controls. These data suggest that MCHeC/eC mice may be protected from aging-associated reductions in p53 protein levels in spleen and liver. It is possible that the increased cancer risk seen in aging animals or humans may potentially be related to aging-induced changes in p53 protein levels. Further studies will be required to gain more insight into this issue. It is also possible that obesity may accelerate aging-associated p53 reduction in a tissue-specific fashion and that protection from aging-associated obesity may retard this process. Although, it is still unknown whether aging-linked reductions in p53 protein levels directly increase cancer risk, attenuation of aging-associated decreases in p53 levels in MCHeC/eC mice is noteworthy and merits further investigation.
In summary, we have confirmed a lean, active, and hypermetabolic phenotype in mice lacking MCH up to 19 months of age in both males and females. In addition, we have shown that although aging increased insulin resistance in wild-type mice, MCHeC/eC mice do not develop insulin resistance as they age. Our results also demonstrate an aging-associated decrease in the tumor suppressor protein p53 in liver and spleen that is attenuated in MCHeC/eC mice. Thus, MCH ablation over the long term appears to mitigate several adverse consequences of the aging process.
ACKNOWLEDGMENTS
E.M.-F. has received National Institute of Diabetes and Digestive and Kidney Diseases Grants PPG-DK-56116 and RO1-DK-56113. J.Y.J. has received a postdoctoral fellowship award from the Natural Science and Engineering Council of Canada. R.L.B. has received National Institute of Diabetes and Digestive and Kidney Diseases Grant KO1-DK-063080.
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2 Research Division, Joslin Diabetes Center, Boston, Massachusetts
3 Department of Medicine, Harvard Medical School, Boston, Massachusetts
4 Department of Sport and Leisure Studies, Yonsei University, Seoul, Korea
5 Division of Gastroenterology, Department of Medicine, Beth Israel Medical Center, Boston, Massachusetts
Key Words: AUC, area under the curve DEXA, dual-energy X-ray absorptiometry GTT, glucose tolerance test ITT, insulin tolerance test MCH, melanin-concentrating hormone REE, resting energy expenditure Sir2, silent inflammatory regulator 2
ABSTRACT
Ablation of the hypothalamic peptide, melanin-concentrating hormone (MCH), leads to a lean phenotype and resistance to diet-induced obesity. Observation of MCHeC/eC mice at older ages suggested that these effects persist in mice >1 year old. Leanness secondary to caloric restriction is known to be associated with improved glucose tolerance as well as an overall increase in life span. Because the MCHeC/eC model represents leanness secondary to increased energy expenditure rather than caloric restriction, we were interested in determining whether this model of leanness would be associated with beneficial metabolic effects at older ages. To assess the effects of MCH ablation over a more prolonged period, we monitored male and female MCHeC/eC mice up to 19 months. The lean phenotype of MCHeC/eC mice persisted over the duration of the study. At 19 months, MCHeC/eC male and female mice weighed 23.4 and 30.8% less than their wild-type counterparts, a result of reduced fat mass in MCHeC/eC mice. Aged MCHeC/eC mice exhibited better glucose tolerance and were more insulin sensitive compared with wild-type controls. Aging-associated decreases in locomotor activity were also attenuated in MCHeC/eC mice. We also evaluated two molecules implicated in the pathophysiology of aging, p53 and silent inflammatory regulator 2 (Sir2). We found that expression of the tumor suppressor protein p53 was higher in MCHeC/eC mice at 9 and 19 months of age. In contrast, expression of Sir2 was unchanged. In aggregate, these findings suggest that MCH ablation improves the long-term outcome for several indicators of the aging process.
Advancing age is associated with increased susceptibility to numerous diseases. A progressive increase in visceral adiposity is a common feature of aging, and epidemiological evidence supports a role of adiposity as a prominent risk factor for metabolic disorders such as insulin resistance, type 2 diabetes, and cardiovascular disease (1eC3). Reversal of obesity through caloric restriction, lifestyle modifications, drug therapy, and surgical removal of visceral fat has beneficial effects on insulin resistance and other metabolic disorders associated with aging (4,5). Similarly, in rodent models, targeted deletion of specific genes has led to reductions in mouse body weights with concomitant improvements in their overall metabolic profiles (6eC8). However, information on the long-term effects of gene deletioneCinduced weight loss on aging-associated metabolic diseases and on other indicators of the aging process is very limited.
Melanin-concentrating hormone (MCH), which is expressed in the lateral hypothalamus, has emerged as an important regulator of energy balance and is also implicated in other behaviors such as water ingestion and anxiety (9eC11). Chronic infusion of MCH leads to excess weight gain and a shift of the animal to a "lipogenic" state associated with decreased energy expenditure and increased energy storage (12,13). When MCH was overexpressed in the lateral hypothalamus of mice, both an increase in food intake and weight gain were observed (14). On the other hand, ablation of both the MCH gene (MCHeC/eC) and the rodent receptor for MCH, designated MCHR-1, leads to a lean phenotype as a consequence of increased locomotor activity and increased basal metabolic rate (6,15). When MCH was genetically ablated, two other neuropeptides, which are derivatives of prepro-MCH and are designated N-EI and N-GE, were also ablated. Neither of these peptides has an effect on feeding alone, in combination with each other, or in combination with MCH (E.M.-F., unpublished data). Although a potential role of these peptides in the lean phenotype of the MCHeC/eC mouse has not been fully excluded, such a role is unlikely because the phenotype of mice lacking the MCH receptor is very similar to that of mice lacking the ligand (15).
Although we have found that MCHeC/eC mice are lean, hyperactive, and hypermetabolic (6,16,17), it is unknown whether this phenotype is maintained as the animals age. Leanness secondary to caloric restriction is known to be associated with improved glucose tolerance as well as with an increased life span. Because MCHeC/eC mice are lean secondary to increased energy expenditure rather than caloric restriction, we sought to determine whether MCH ablation would result in improved long-term outcomes for adiposity and insulin sensitivity, as well as for other indicators of the aging process. Therefore, we evaluated the phenotype of MCHeC/eC mice up to 19 months old. Parameters assessed included food intake, body weight, locomotor activity, and insulin sensitivity. In addition, we initiated an evaluation of potential long-term effects of MCH ablation on two key molecular markers linked to cellular senescence and organismal aging, namely the tumor suppressor protein p53 and the silent inflammatory regulator 2 (Sir2) protein, a member of the conserved sirtuin family of nicotinamide adenine dinucleotideeCdependent deacetylases (18,19). p53 is an important regulator of cell growth, apoptosis, and DNA repair (20,21), and numerous lines of evidence indicate that p53 plays divergent roles in the aging process (22eC24). It has also been reported that p53-deficient mice, which die at relatively young ages because of malignancies, show accelerated age-related accumulation of mutations in spleen and liver tissues (25). With regard to the role of Sir2 in aging, numerous studies in yeast and Drosophila have shown that caloric restriction increases longevity at least in part by increasing the activity and expression of Sir2 protein (26eC28). A recent report suggested that Sir2 homologs may also be important for longevity in humans (29). Furthermore, a human Sir2 homolog has been shown to bind to p53 and to downregulate p53-induced apoptosis (30,31). Therefore, we measured Sir2 and p53 protein levels in liver and spleen tissues from 9- and 19-month-old MCHeC/eC mice and their wild-type littermates, with the hypothesis that an attenuated aging phenotype in lean MCHeC/eC mice might be mediated in part via effects on these proteins.
RESEARCH DESIGN AND METHODS
These studies were approved by the Joslin Diabetes Center Animal Care and Use Committee. Mice lacking the gene for prepro-MCH were generated as previously described (6). For the current studies, MCHeC/eC mice were backcrossed onto a C57BL/6 background for eight generations. Mice were housed up to four per cage in the Joslin Animal Facility and maintained at 22°C, under an alternating 12-h light/dark cycle. Mice were placed on a normal diet (Purina Lab Diet 5008; 3.5 kcal/g) and allowed access to food and water ad libitum. Animals were monitored over a period of 19 months.
Glucose tolerance tests.
Intraperitoneal glucose tolerance tests (GTTs) were performed at the ages of 10 and 16 months. Mice were fasted overnight (1700eC0800) and were subsequently injected with glucose (2 g/kg body wt i.p.). Tail blood was collected at eC1, 15, 30, 60, and 120 min. Blood glucose concentrations were measured using a glucometer (Elite; Bayer, Mishawaka, IN).
Insulin tolerance tests.
Insulin tolerance tests (ITTs) were performed at the ages of 10 and 16 months. Mice were fasted for 3 h and subsequently injected with regular insulin (1 unit/kg; Eli Lilly and Co., Indianapolis, IN). Tail-blood samples were collected just before insulin administration and at 15, 30, 60, and 120 min after the insulin injection.
Oxygen consumption.
Oxygen consumption of 6- and 18-month-old mice was measured in eight open-circuit Oxymax chambers that are a component of the Comprehensive Laboratory Animal Monitoring System (Columbus Instruments, Columbus, OH). Mice were housed individually and were maintained at 24°C under a 12-h light/dark cycle (light period 0800eC2000) with free access to food and water. All mice were acclimatized to monitoring cages for 24 h before beginning an additional 24 h of hourly recordings of physiological parameters.
Resting energy expenditure.
Resting energy expenditure (REE) was determined by defining periods of inactivity and measuring oxygen consumption during those periods. Intervals during which animals showed <50 beam breaks were considered inactive intervals.
Locomotor activity.
Ambulatory activity of individually housed mice was evaluated on a relative basis using an eight-cage rack OPTO-M3 Sensor system (Columbus Instruments). Consecutive photobeam breaks occurring in adjacent photobeams were scored as an ambulatory movement. Cumulative ambulatory activity counts were recorded every hour for 24 h.
Body composition.
Fat and lean body mass were assessed using dual-energy X-ray absorptiometry (DEXA; Lunar PIXImus2 mouse densitometer; GE Medical Systems, Madison, WI) as described by the manufacturer. Mice were anesthetized by intraperitoneal injection of a 1:1 mixture of tribromoethanol:tert-amyl alcohol (0.015 ml/g body wt) and scanned, and total body fat and lean body mass were determined using an analysis program provided by the manufacturer.
Immunoblotting.
Liver and spleen tissues were homogenized in lysis buffer (20 mmol/l Tris-Cl, pH 7.5, 150 mmol/l NaCl, 1 mmol/l EDTA, and 1% Triton X-100) supplemented with various protease inhibitors. Protein concentration of the extracts was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total protein (25 e蘥) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with a mouse monoclonal -p53 antibody (Abcam, Cambridge, MA) or with a rabbit polyclonal -Sir2 antibody (Upstate USA, Lake Placid, NY). Signals were visualized using the Super Signal West Pico chemiluminescence reagent (Pierce, Rockford, IL).
Statistics.
Values are reported as group means ± SE. Interactions between genotype and physiological parameters were analyzed by two-way ANOVA. One-way ANOVA and independent t test were also used where appropriate. A P value of <0.05 was considered statistically significant. Statistical comparisons were made using Statview software (Abacus Concepts, Berkley, CA).
RESULTS
MCHeC/eC mice have a lean phenotype because of increased physical activity and energy expenditure.
Growth curves showed that MCH-ablated mice had a marked attenuation of weight gain normally associated with aging in both males and females (Fig. 1A and B). At 19 months of age, male MCHeC/eC mice weighed 32.6 ± 1.5 g, and male wild-type mice weighed 42.9 ± 1.5 g. Similarly, female MCHeC/eC mice weighed 31.2 ± 3.1 g, and female wild-type mice weighed 45.1 ± 2.6 g (P < 0.05). Body fat measurement by DEXA analysis showed that most of the weight difference was due to reduced fat mass (Fig. 1C and D). Dissection of MCHeC/eC mice revealed a marked reduction in perigonadal white adipose tissue (Fig. 1E) (MCHeC/eC males, 807 ± 171; wild-type males, 2,943 ± 281; MCHeC/eC females, 1,381 ± 879 mg; and wild-type females, 6,039 ± 1,108 mg; P < 0.05). The weight of other major organs such as liver (Fig. 1F) was not affected after correcting for total body weight.
The attenuated body weight gain in MCHeC/eC mice was not due to reduced feeding, because there was no significant difference in total food intake (food intake at 18 months; Fig. 1G and H). Food intake was also measured at 2, 7, and 10 months, and no significant difference in food intake was seen at any of these time points (not shown). This finding confirms a previous report from our laboratory showing no difference in food intake in younger male MCHeC/eC and male wild-type mice on a pure C57BL/6 background (32). Leanness in MCHeC/eC mice was observed at both young and old ages and was secondary to increases in both locomotor activity and basal metabolic rate (Fig. 2A and B). Analysis of locomotor activity showed that older wild-type mice had significantly reduced locomotor activity compared with younger mice. However, MCHeC/eC mice maintained increased locomotor activity at older ages (Fig. 2C) and displayed a resting metabolic rate comparable with younger animals. In addition, REE was reduced with aging by 11% in wild-type mice, whereas older MCHeC/eC mice had REE levels comparable with those of younger MCHeC/eC mice (Fig. 2D).
MCHeC/eC mice are resistant to aging-associated glucose intolerance.
A series of GTTs and ITTs were performed on both male and female MCHeC/eC mice at different ages. There was no difference in glucose tolerance in either male or female MCHeC/eC mice compared with their wild-type counterparts when they were 2 months old. Improved glucose tolerance was observed in male MCHeC/eC mice at 10 months of age when these animals showed a 28% reduction in glucose area under the curve (AUC) during GTTs compared with wild-type littermates (Fig. 3A). This difference in glucose tolerance in males became even more evident as they continued to age. As shown in Fig. 3B, at 16 months of age, male wild-type mice had 41.7% higher glucose AUC compared with their MCHeC/eC counterparts. At 10 months, female MCHeC/eC mice had similar glucose tolerance as their wild-type counterparts (Fig. 3C). However, a marked difference in glucose tolerance also manifested itself in female MCHeC/eC mice compared with their wild-type counterparts at 16 months. As shown in Fig. 3D, at this age, female wild-type mice had 108% higher glucose AUC compared with female MCHeC/eC mice. Two-way ANOVA repeated measurements showed that both male and female wild-type mice had significantly worse glucose tolerance than MCHeC/eC mice (P = 0.01). In addition, ITTs revealed that wild-type male mice were more insulin resistant compared with their MCHeC/eC counterparts (Fig. 3E and F). Plasma insulin levels during GTTs were significantly lower in 16-month-old MCHeC/eC mice compared with wild-type controls (Fig. 3G). At 16 months of age, consistent with their better insulin sensitivity, MCHeC/eC mice had substantially lower fasting insulin levels compared with wild-type animals. This effect was seen in both male and female animals (Fig. 4A and B).
MCHeC/eC mice show attenuation of tissue-specific aging-associated decreases in the tumor suppressor protein p53.
To begin defining potential anti-aging effects of MCH ablation at a molecular level, we measured Sir2 and p53 levels in liver and spleen tissues by immunoblotting. No significant difference in Sir2 protein levels was seen at any age between wild-type and MCHeC/eC mice in either liver or spleen (not shown). In contrast, as shown in Fig. 5A and B, aging was associated with a decline in p53 levels that was seen in both 19-month-old wild-type and MCHeC/eC mice, compared with 9-month-old animals. This effect was noted in both liver (9-month-old wild type, 1 ± 0.38; 9-month-old MCHeC/eC, 1.58 ± 0.17; 19-month-old wild type, 0.38 ± 0.08; and 19-month-old MCHeC/eC, 1.28 ± 0.21; P < 0.05) and spleen (9-month-old wild type, 1 ± 0.04; 9-month-old MCHeC/eC, 2.23 ± 0.4; 19-month-old wild type, 0.5 ± 0.2; and 19-month-old MCHeC/eC, 1.30 ± 0.2; P < 0.05). However, the progressive decline in p53 levels was attenuated in MCHeC/eC mice compared with their wild-type counterparts.
DISCUSSION
We previously reported that MCHeC/eC mice on a mixed genetic background (C57BL/6 and 129) were lean and hypophagic (6). Here, we have studied mice backcrossed eight generations onto a C57BL/6 background and monitored up to 19 months of age. On this background, MCHeC/eC mice are lean, hyperactive, and insulin sensitive compared with their wild-type littermates up to 19 months. As previously reported in young C57BL/6 mice lacking MCH, the lean phenotype was not due to a reduction in food intake. Measurements of food intake showed that MCHeC/eC mice had similar food intake to their wild-type counterparts. Reductions in body weight in MCHeC/eC mice on a C57BL/6 background were secondary to increased energy expenditure, resulting from both increased locomotor activity and basal metabolic rate. In MCHeC/eC mice, both were sustained at higher levels at older ages, while progressively declining in wild-type mice; however, the neuroendocrine systems mediating these changes downstream of MCH neurons remain unknown.
The increase in resting metabolic rate in MCHeC/eC mice may be due to increased thermogenesis, which in turn may be a consequence of increased sympathetic nervous system activity. We have previously found higher uncoupling protein-1 levels in brown adipose tissue from MCHeC/eC mice compared with their wild-type counterparts (16). Furthermore, mice lacking MCHR-1 also show a hypermetabolic phenotype due to increased autonomic activity (15,33).
A progressive increase in visceral adiposity is a common feature of aging (1,34). Increased visceral fat contributes to increased insulin resistance and reversal of visceral obesity via caloric restriction and/or surgical removal of visceral fat increases insulin sensitivity (35,36). In the current study, we found that 19-month-old MCHeC/eC mice did not develop glucose intolerance and maintained glucose sensitivity comparable with young wild-type mice. Because the amount of fat in MCHeC/eC mice was markedly reduced in both males and females compared with wild-type mice, we hypothesize that the more sensitive glucose tolerance phenotype seen in MCHeC/eC mice may be due to reduced visceral fat mass. Increased visceral adiposity with aging has been observed in animals and in humans, and surgical removal of visceral fat in 20-month-old rats was sufficient to restore peripheral and hepatic insulin action to levels seen in young rats (5,34,35). Therefore, the lack of visceral adiposity in older MCHeC/eC mice may explain the preservation of glucose tolerance and insulin sensitivity seen in these animals.
In mammals, caloric restriction delays the onset of aging-associated disorders, including cancer, atherosclerosis, and diabetes and can greatly increase life span (37eC41). As described earlier, both Sir2 and p53 are implicated in aging at the cellular and organismal levels. Sir2 genes play a central role in evolutionarily conserved pathways of aging and longevity. In yeast and Caenorhabditis elegans, life span is extended by the presence of extra copies of the SIR2/Sir-2.1 genes (42). However, little is known regarding the role of Sir2 in mammalian aging. Therefore, we measured Sir2 protein levels in 9- and 19-month-old MCHeC/eC mice and their wild-type littermates. We evaluated liver and spleen tissues from these animals, as we were examining Sir2 protein levels in tandem with p53 protein levels in these tissues on the basis that Sir2 is a negative regulator of p53 activity and p53-null mice are reported to have accelerated age-related occurrences of mutations in these two organs (25). Furthermore, the liver represents a primary target organ for obesity- and insulin resistanceeCinduced metabolic and inflammatory changes. In contrast to a report of increased Sir2 protein expression in long-term calorie-restricted rats (18), there was no difference in Sir2 protein expression between MCHeC/eC mice and wild-type mice, suggesting that leanness in MCHeC/eC mice does not upregulate Sir2 protein expression.
Advancing age is also associated with an increased risk of cancer and cellular senescence in response to oncogenic signals (43). In humans, the incidence of cancer rises exponentially in the final decades of life, resulting in a cumulative life time risk of one in two for men and one in three for women (43). As previously discussed, the p53 gene encodes a potent tumor suppressor protein, which induces cell cycle arrest and apoptosis and is implicated as a mediator of organismal aging as well. Results to date indicate that p53 protein levels are differentially regulated in a tissue-specific pattern during aging. For example, some studies have shown increased p53 levels in the gastric mucosa of rats aged up to 24 months (44), whereas others have shown a decline in p53 levels in the colonic mucosa (45) or no change in myocyte p53 levels in rats aged up to 24 months (46). Interestingly, we observed in both wild-type and MCHeC/eC mice that p53 protein levels in spleen and liver were substantially decreased at 19 months compared with at 9 months. To our knowledge, this is the first report showing an aging-associated decrease in p53 protein levels in mouse liver and spleen. However, aged MCHeC/eC mice still retained higher p53 levels in both liver and spleen than wild-type controls. These data suggest that MCHeC/eC mice may be protected from aging-associated reductions in p53 protein levels in spleen and liver. It is possible that the increased cancer risk seen in aging animals or humans may potentially be related to aging-induced changes in p53 protein levels. Further studies will be required to gain more insight into this issue. It is also possible that obesity may accelerate aging-associated p53 reduction in a tissue-specific fashion and that protection from aging-associated obesity may retard this process. Although, it is still unknown whether aging-linked reductions in p53 protein levels directly increase cancer risk, attenuation of aging-associated decreases in p53 levels in MCHeC/eC mice is noteworthy and merits further investigation.
In summary, we have confirmed a lean, active, and hypermetabolic phenotype in mice lacking MCH up to 19 months of age in both males and females. In addition, we have shown that although aging increased insulin resistance in wild-type mice, MCHeC/eC mice do not develop insulin resistance as they age. Our results also demonstrate an aging-associated decrease in the tumor suppressor protein p53 in liver and spleen that is attenuated in MCHeC/eC mice. Thus, MCH ablation over the long term appears to mitigate several adverse consequences of the aging process.
ACKNOWLEDGMENTS
E.M.-F. has received National Institute of Diabetes and Digestive and Kidney Diseases Grants PPG-DK-56116 and RO1-DK-56113. J.Y.J. has received a postdoctoral fellowship award from the Natural Science and Engineering Council of Canada. R.L.B. has received National Institute of Diabetes and Digestive and Kidney Diseases Grant KO1-DK-063080.
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