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Department of Medicine, Banting and Best Diabetes Center, Toronto Hospital, University of Toronto, Toronto, Ontario, Canada M5G 2C4
Address all correspondence and requests for reprints to: Dr. D. Drucker, Toronto Hospital, 200 Elizabeth Street, CCRW3–838, Toronto, Ontario, Canada M5G 2C4. E-mail: d.drucker{at}utoronto.ca
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Abstract |
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Introduction |
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GLP-1 is also synthesized in the central nervous system (CNS), predominantly in the brain stem and hypothalamus (16), and brain stem projections have been identified that appear to transport GLP-1 to diverse regions of the CNS (17, 18). The initial observation that GLP-1 activated adenylate cyclase activity in hypothalamic and pituitary membrane preparations was followed by studies demonstrating GLP-1-binding sites and GLP-1R expression in different brain regions (19, 20); however, the precise physiological role(s) for GLP-1 in the CNS remained unclear. The demonstration that GLP-1 was a potent inhibitor of food intake coupled with the high density of GLP-1-binding sites in hypothalamic nuclei important for control of satiety strongly suggested that GLP-1 was an important peptide mediator of feeding behavior and weight control (21). Furthermore, GLP-1 inhibited the neuropeptide Y (NPY)-induced stimulation of food intake and blockade of CNS GLP-1Rs with the antagonist exendin-(9–39) potentiated the NPY-induced stimulation of feeding (21). These experiments provided important new evidence implicating the GLP-1R as a key mediator of peptidergic regulation of feeding in vivo.
Molecular cloning of GLP-1Rs from pancreatic islets, heart, lung, and brain (22, 23, 24, 25) has provided direct evidence that the amino acid sequence of the GLP-1R is identical in all of these tissues. Accordingly, derivation of mice with a complete disruption of GLP-1R signaling provides a useful opportunity to assess the phenotypic consequences of GLP-1R deficiency in both the CNS and peripheral tissues in vivo. GLP-1R-/- mice exhibit fasting hyperglycemia and abnormal glycemic excursions after both oral and ip glucose challenge (26). These abnormalities together with reduced levels of glucose-stimulated insulin (26) define an essential role for GLP-1 in the control of blood glucose. Surprisingly, despite complete elimination of GLP-1-binding sites in the hypothalamus, GLP-1R-/- mice are not obese and do not exhibit abnormalities in feeding behavior (26). Nevertheless, the initial characterization of glucose tolerance and feeding behavior in GLP-1R-/- mice was carried out on young mice, 8–10 weeks of age. Accordingly, it remained possible that analysis of older animals might reveal additional defects in glucose homeostasis and possibly abnormalities in satiety control or weight regulation. We now report the results of studies examining the response to high fat feeding and the effects of aging in GLP-1R-/- mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aging studies. Male and female wild-type (+/+), heterozygote (+/-), and homozygote (-/-) littermates derived from matings of mice heterozygous for the GLP-IR (genotype confirmed by Southern blotting) were placed into separate cages at weaning (n = 5/group) under normal housing conditions with a 12-h light, 12-h dark cycle and ad libitum access to water and rodent chow. All mice were weighed at 11 months of age and again at the end of the experiment at 16 months of age.
High fat studies. For high fat feeding studies, 12-week-old wild-type CD1 and GLP-IR -/- mice (n = 30/group) were provided ad libitum access for 18 weeks to a defined rodent chow (D12451) containing 45% of the total calories from fat (Research Diets, New Brunswick, NJ). At all other times mice received Lab Diet 5001, containing 4.5% of the total calories from fat (PMI Feeds, St. Louis, MO). Once each week the mice were weighed, and the amount of chow consumed was recorded. After 18 weeks of high fat diet, oral glucose tolerance tests (OGTTs) were performed on (now 30-week-old) wild-type and GLP-IR -/- mice (n = 5/group) as described above.
Glucose and insulin determinations
For assessment of glucose tolerance by OGTT, mice were fasted
overnight for 18 h. OGTT was carried out by administering 1.5 mg
glucose/g BW orally through a gavage tube. Blood was withdrawn from a
tail vein at 0, 10, 20, 30, 60, 90, and 120 min, and blood glucose
levels were measured with a ONE TOUCH BASIC glucose monitor (Lifescan
Canada, Delta, Canada). Plasma insulin levels were determined in
duplicate using an insulin RIA kit (Linco, St. Charles, MO) with rat
insulin as a standard. Values are the mean ± SEM.
Blood for insulin determinations was collected by tail bleeds induced
by a 1- to 2-mm tail nick from minimally restrained, hand-held mice.
The blood was collected 20–30 min after glucose administration into
5000 kallikrein inhibitory units/ml Trasylol-32 mM EDTA-0.1
nM diprotin A; plasma was separated by centrifugation and
stored at -80 C until assayed. Statistical analysis was performed
using the InStat program (Graph Pad Software, San Diego, CA) for the
Macintosh. All animal studies were carried out in accordance with
guidelines and protocols approved by the Toronto Hospital animal care
committee.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The identical groups
of mice were subsequently maintained on normal rodent chow for a
further 5 months. Although a few 16-month-old
GLP-1R-/- females were heavier than wild-type
controls, this difference was not statistically significant (Fig. 1A).
Furthermore, no difference in weights between 16-month-old control and
GLP-1R-/- male mice was detected. These
observations demonstrate that unlike other rodent models of obesity
(29), GLP-1R-/- mice do not develop increased
weight gain with advancing age.
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). Glucose tolerance was
impaired in 16-month-old female GLP-1R-/- mice,
and the difference in blood sugars (compared with age-matched wild-type
controls) was statistically significant (P <
0.05–0.01). In contrast to the abnormal glucose tolerance previously
demonstrated in 6- to 8-week-old male GLP-1R-/-
mice (26), glucose tolerance was not significantly different (from
controls) in 16-month-old male GLP-1R-/- mice
(Fig. 1B). Furthermore, we did not detect a significant difference in
the levels of glucose-stimulated insulin in 16-month-old
GLP-1R-/- mice (Fig. 1C). Taken together, these
experiments clearly demonstrate that the phenotype of glucose
intolerance does not markedly deteriorate and may even improve
(relative to that in age-matched wild-type controls) in older mice with
disruption of GLP-1 signaling.
To determine the effects of high fat feeding on body weight and glucose
control in the absence of central or peripheral GLP-1 action, control
and GLP-1R-/- mice were maintained for 18 weeks
on a high fat rodent diet (45% of the total calories from fat). As
shown in Fig. 2A, although both wild-type
and GLP-IR-/- female mice gained weight on the
high fat diet, wild-type mice gained significantly more weight (31%
increase from 35.5 ± 0.5 to 46.4 ± 1.3 g) than did
female GLP-IR -/- mice (18% increase from
29.3 ± 0.9 to 34.4 ± 1.8 g; P <
0.001, control vs. GLP-1R-/- mice)
from 10–18 weeks. In contrast, the weights of male wild-type control
CD1 mice increased by 28%, whereas the weights of
GLP-1R-/- mice consuming the identical high fat
diet increased by 25% (Fig. 2A; P = NS).
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) maintained on either
regular rodent or high fat chow for 18 weeks. Although blood
glucose excursion was slightly greater in
GLP-1R-/- mice maintained on a high fat diet,
this difference was not statistically significant (Fig. 2B). In
contrast, after high fat feeding, the levels of circulating insulin
after glucose challenge were markedly elevated in
GLP-1R-/- mice (Fig. 2C). These observations
raise the possibility of a differential induction of insulin resistance
and/or ß-cell sensitivity in response to the high fat feeding,
possibly to fatty acids, in GLP-1R-/- mice that
merits further investigation. |
Discussion |
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Although both leptin and GLP-1 inhibit food intake, comparison of the inhibitory effects of leptin vs. GLP-1 revealed several important differences. First, analysis of c-Fos-like immunoreactivity in the brains of peptide-injected rats demonstrated differential stimulation of fos activation in different brain nuclei by these two peptides, implying unique pathways for the actions of leptin and GLP-1 (34). Furthermore, whereas both leptin and GLP-1 inhibit feeding in short term studies, analysis of 16-h food consumption and 24-h changes in body weight after icv peptide administration demonstrated that only the inhibitory effects of leptin, but not GLP-1, were significant at these longer time points (31). These observations together with experimental data demonstrating that GLP-1 inhibits short term, but not long term, food intake in lean and obese rats (35) suggest that the inhibitory effects of GLP-1 on food intake may be relatively transient. These temporal differences between GLP-1 and leptin on suppression of food intake may explain why disruption of leptin signaling is associated with marked changes in food intake and body weight (36), whereas disruption of GLP-1 signaling, as shown here, produces no long term detectable phenotypic changes in food intake or body weight (26).
As obesity commonly arises secondary to a mismatch between caloric intake and expenditure, we hypothesized that exposure of GLP-1R-/- mice to a high fat diet might predispose the mice to the development of obesity. No difference in weight gain was observed in male wild-type vs. GLP-1R-/- mice, and remarkably, female GLP-1R-/- mice gained significantly less weight, compared with wild-type controls, after 18 weeks of high fat feeding. Despite exposure to a high fat diet for 18 weeks, control CD-1 mice did not develop obesity or glucose intolerance. The murine response to high fat feeding and susceptibility to the development of obesity appear to be highly strain specific (37). The failure of GLP-1R-/- mice to develop obesity may therefore be related in part to the CD-1 genetic background that harbors the GLP-1R mutation. Alternatively, multiple compensatory changes in the factors that regulate appetite control and body weight might mitigate against increased body weight in the absence of hypothalamic GLP-1 signaling.
Evidence of the existence of compensatory mechanisms in the control of food intake derives from studies of mice with NPY deficiency. Despite the central role of neuropeptide Y in the stimulatory control of food intake, mice with disruption of the NPY gene do not exhibit disturbances of feeding or body weight (38); however, mice with mutations at both the ob and NPY loci eat less, have increased energy expenditure, and are significantly less obese than ob/ob mice with normal NPY function (39). These observations together with the demonstration that NPY-deficient mice are leptin sensitive (38) demonstrate important functional interactions among peptidergic networks that control body weight and raise the possibility that redundancy at the level of CNS GLP-1 signaling might compensate for the lack of inhibitory GLP-1 action on feeding behavior in vivo.
Intriguingly, we observed a gender-specific difference in the response to high fat feeding, with GLP-1R-/- female mice gaining comparatively less weight than their male counterparts. Studies of heterozygous mice with a mutation in one glucokinase allele also demonstrated that female mice gained less weight on a high fat diet (40). We previously observed that younger male GLP-1R-/- mice exhibited a greater degree of glucose intolerance than age-matched female littermates (26). The mechanism(s) responsible for these gender-specific differences remains unknown; however, gender-specific differences in murine diabetes are not uncommon. For example, the cumulative incidence of diabetes in the NSY mouse is 98% in males and 31% in females at 48 weeks of age (41). Similarly, a greater degree of glucose intolerance has been observed in male transgenic mice of different genetic backgrounds in several different studies (42, 43).
Surprisingly, despite previous observations that glucose intolerance in rodents often deteriorates with increasing age (41), we did not detect worsening of oral glucose tolerance in older GLP-1R-/- mice. The glycemic excursion after oral glucose challenge was actually similar in 16-month-old male wild-type CD1 and GLP-1R-/- mice. These results may be explained in part by a mild deterioration in glucose excursion with aging in older CD1 control mice. Furthermore, despite the well known deleterious effects of increased fatty acids on insulin resistance and insulin secretion (44), glucose tolerance did not deteriorate in GLP-1R-/- mice consuming a high fat diet. The reason for the markedly increased glucose-stimulated insulin levels in GLP-1R-/- mice after high fat feeding remains unknown, but may reflect the need of GLP-1 R-/- islets to overcome peripheral insulin resistance through increased insulin secretion. Comparison of the glucose-stimulated insulin levels in 16-month-old mice reported here vs. levels in 8-week-old GLP-1R-/- characterized previously (26) demonstrates that glucose-stimulated insulin levels are actually higher in the older GLP-1R-/- mice, consistent with the relatively normal glycemic excursion detected in older GLP-1R-/- mice. These observations highlight the importance of longitudinal studies in the phenotypic assessment of mouse models of diabetes, as metabolic abnormalities detected in younger mice may be subject to modification as the animal ages. Taken together, the demonstration that aging and high fat feeding do not induce obesity in GLP-1R-/- mice suggests that either GLP-1 is not essential for regulation of body weight or multiple compensatory mechanisms exist for the control of feeding and body weight in vivo.
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Acknowledgments |
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Footnotes |
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Received December 17, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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