GI of the test meals
After the training period, six of eight animals in each group consumed the entire test meals. Differences for glucose and insulin at single time points are shown in Fig. 1a, b.
Body weight gain only in younger adult mice on high- vs low-GI diets
Body weight at baseline was significantly lower in the young-adult vs aged mouse cohorts (p < 0.001), as expected. The influence of the diet on the time course of increases in body weight was significant in young-adult (p < 0.001; Fig. 2a), but not in aged mice (p = 0.12; Fig. 2b). In both young and aged animals the Gipr
−/− genotype had no significant effect on changes in body weight (p > 0.55). When comparing the 20 week intervention period of young-adult mice with the first 20 week period in aged animals the interaction of time × diet × age showed a significant influence (p < 0.001). However, differences in body weight between groups in aged mice did not reach the significance level, although we extended the intervention period in aged animals by a further 6 weeks (26 vs 20 weeks, compared with the younger mouse cohort). Therefore, the significant influence of the high-GI diet on body weight appeared to be age-dependent.
Increased body fat and hepatic triacylglycerol content on high-GI diet
The diet significantly influenced changes in body fat both in young-adult mice after 9 and 16 weeks (two-way ANOVA, p < 0.001; Fig. 2c) and in aged mice after 8 and 24 weeks (p < 0.05; Fig. 2e) of dietary intervention. However, in subgroup analyses, significant differences in body fat between groups were only detected in young-adult mice (wild-type, high- vs low-GI diet, after 9 and 16 weeks, p < 0.01; Gipr
−/−, high- vs low-GI diet, after 9 and 16 weeks, p < 0.05), but not in aged mice (Fig. 2e, p > 0.05, respectively). There was no effect of the Gipr genotype on body fat in young-adult and in aged mice (p = 0.69 and p = 0.26, respectively). Relative changes in body fat showed similar results, with a significant effect of the diet at week 16 in young-adult (p < 0.001) and week 24 in aged (p = 0.013) mice. Two-way ANOVA analysis of lean mass alteration in young wild-type mice on high- and low-GI and Gipr
−/− on high- and low-GI diets (21.1 ± 0.3, 21.0 ± 0.3, 21.2 ± 0.3 and 21.1 ± 0.3 g) was influenced neither by the genotype (p = 0.81) nor by diet (p = 0.92) nor diet × genotype interaction (p = 0.98) after 16 weeks. There was also no influence of these three factors in aged mice after 24 weeks (21.5 ± 0.5, 20.8 ± 0.2, 20.6 ± 0.3 and 21.5 ± 0.4 g, with p = 0.81, p = 0.731 and p = 0.08, respectively).
In agreement with in vivo body fat nuclear magnetic resonance analysis, hepatic triacylglycerol content was significantly increased in both young (Fig. 2d, p < 0.001) and aged animals (Fig. 2f, p = 0.02) fed the high-GI diet, with no influence of the Gipr
−/− genotype (p > 0.46).
Higher energy digestion with high- vs low-GI diets, regardless of age and Gipr genotype
Digestibility of diets (%) was significantly elevated in high- vs low-GI fed wild-type animals, in both younger (95.3 ± 0.2 vs 90.8 ± 0.4, p < 0.001) and aged (94.7 ± 0.3 vs 89.5 ± 0.9, p < 0.001) mice. Similar differences were observed in younger (95.2 ± 0.3 vs 90.1 ± 0.4, p < 0.001) and aged (94.8 ± 0.6 vs 90.8 ± 0.5, p < 0.001) Gipr
−/− mice. There was no difference in energy digestion between younger and aged mice.
Gipr−/− prevents an increase in cumulative energy intake on high-GI diet in aged animals
In young-adult mice, cumulative net energy intake measured by recording of food intake over 18 weeks was not significantly different between groups, regardless of the diet and the Gipr genotype (Fig. 2g). In aged mice, however, cumulative net energy intake was significantly influenced by the diet after 11 (p = 0.006) and 22 weeks (p = 0.015), with a significant interaction of genotype × diet (p = 0.031) after 22 weeks, indicating an increased cumulative net energy intake on high-GI diet only in wild-type aged mice (Fig. 2h).
Improved estimated insulin sensitivity in aged Gipr−/− mice on high-GI diet
The response to i.p insulin injection was tested in all groups of mice after 17 weeks of dietary intervention. No differences in estimated insulin sensitivity were observed in young-adult mice using this method (Fig. 3a), despite the observed diet-induced significant differences in body fat. In aged animals, there was also no difference between Gipr genotypes on low-GI diet (Fig. 3b). However, the observed significant influence of the genotype (p = 0.014) and the interaction of genotype × diet (p = 0.0019) in AUCglucose analysis (plasma glucose suppression by i.p. insulin injection) indicated a protective effect of GIPR deficiency in aged animals on a high-GI diet.
Two-way ANOVA analysis of changes in HOMA-IR in wild-type mice on high- and low-GI diets (7.2 ± 2.0 vs 5.0 ± 1.4) and in Gipr
−/− mice on high- and low-GI diets (3.0 ± 0.8 vs 3.5 ± 0.6) supported a significant influence of the genotype (p = 0.035) in aged but not in young-adult animals (6.9 ± 1.3 vs 4.7 ± 1.7, and 3.5 ± 1.0 vs 3.5 ± 0.8, p = 0.1), whereas diet and the interaction of diet × genotype did not reach a significant level, either in young-adult (p > 0.39) or in aged mice (p > 0.29).
GTTs were additionally performed and used as a further marker for the investigation of insulin resistance. While glucose excursions in young mice were mainly influenced by the high-GI diet (p = 0.004), independently of the Gipr
−/− genotype, in aged mice there was a significantly reduced AUCglucose in high-GI fed Gipr
−/− vs wild-type animals (p = 0.003). These findings were in accord with the results received after i.p. insulin injection, although the interaction of the Gipr genotype with the diet failed to reach the level of significance (p = 0.26). AUCinsulin following i.p. glucose injection was not different on high- vs low-GI diet in all groups of mice (data not shown).
Improved carbohydrate oxidation in aged Gipr−/− mice on high-GI diet
Results from indirect calorimetry further indicated an influence of dietary GI on metabolism in aged Gipr
−/− animals. The RQ in aged high- vs low-GI fed Gipr
−/− mice was significantly higher during the dark phase, where most of the dietary intake takes place (Fig. 3f), indicating improved carbohydrate metabolism. This result was in agreement with the observed shift to improved insulin sensitivity. In contrast, the mirrored pattern of RQ, as observed in younger Gipr
−/− mice, suggested an improved rather than reduced carbohydrate metabolism on low-GI diet (Fig. 3e). Mean RQ showed no significant differences in high- vs low-GI fed wild-type animals, either in young-adult (p = 0.35) or in aged (p = 0.37) mice (data not shown). These data indicated that, in the GIPR-deficient state, the age of the animals contributed to diverse outcomes when assessing potential beneficial effects of low- vs high-GI diets.
Mean energy expenditure per metabolic mass in young wild-type mice on high- and low-GI and Gipr
−/− on high- and low-GI diets (750.8 ± 0.4, 703.6 ± 18.2 and 735.9 ± 11.2, 716.2 ± 22.5 kJ day−1 kg−0.75) was not influenced by genotype (p = 0.95), diet (p = 0.08) or diet × genotype interaction (p = 0.47) in two-way ANOVA analysis. There was also no influence of these three factors in aged animals (574.3 ± 12.3, 583.9 ± 24.7 and 592.0 ± 16.0, 602.5 ± 9.5 kJ day−1 kg−0.75, with p = 0.279, p = 0.54 and p = 0.98, respectively).
Increased locomotor activity in aged Gipr−/− mice on high-GI diet
Given that a significant difference in estimated insulin sensitivity was detected in aged high-GI fed Gipr
−/− vs wild-type mice despite unaltered body weight after 24 weeks of dietary intervention, we hypothesised that potential changes in locomotor activity could have contributed to the observed findings. Therefore, spontaneous locomotor activity was recorded in aged mice after 24 weeks of dietary intervention (Fig. 4).
Spontaneous locomotor activity during the dark phase, where most of the natural activity takes place in rodents, showed a significant influence of the genotype (two-way ANOVA, p < 0.05) and a trend in interaction of diet × genotype (p = 0.09), indicating increased activity levels in the high-GI fed Gipr
−/− vs wild-type animals. No differences between groups were observed during the light phase (Fig. 4).
Changes in hypothalamic orexigenic and anorexigenic factors in aged mice
Two-way ANOVA analysis of mRNA expression of hypothalamic factors related to energy intake revealed a significant genotype × diet interaction in aged mice for Agrp transcription (p = 0.022), and an influence of the diet on Pomc transcription (p < 0.046). Npy and Cart were not significantly influenced by either factor (Fig. 5). Unexpectedly, the anorexigenic factor genes Pomc and Cart in both high-GI fed wild-type and Gipr
−/− animals even tended to increase, probably representing a compensatory mechanism. The orexigenic Agrp tended to decrease only in Gipr
−/− mice on low-GI diet (subgroup analysis, Fig. 5), while Npy showed no obvious tendencies between groups.