This study examined the effects of a mid life (11 months of age) dietary switch from 30% DR to AL (DR-AL) feeding or from AL feeding to 30% DR (AL-DR) on a range of metabolic parameters and compared these to animals maintained exclusively on AL feeding or 30% DR from early life (3 months of age). In particular we were interested in whether the experience of early-life DR could confer metabolic benefits in mice subsequently switched back to an AL diet, that is, does a metabolic memory exist? Currently, there is very little experimental evidence to suggest that mice retain a metabolic memory to previous DR exposure. A dietary shift from AL-DR in adult flies [25, 26], rats  and mice  resulted in animals rapidly adopting the decreased mortality risk of long-term DR animals. The converse shift in flies [25, 26] and rats  has also been shown to increase the mortality risk to that of AL animals. In addition, the shift from DR-AL rapidly reverses the majority of the transcriptional changes induced by long-term DR [17, 18]. In contrast to the studies above [25–27], a dietary shift from DR-AL in rats demonstrated a benefit to early-life DR on lifespan , that is, the DR-AL rats lived significantly longer than animals maintained on AL feeding throughout their life. Similar evidence of a metabolic memory to previous DR exposure was also reported in a second rat study . Interestingly, this study showed that those animals switched from DR-AL (or AL-DR) at either 6 months or 12 months only retained the survival trajectory of the initial feeding regime when they were additionally dietary supplemented with α-lipoic acid .
In terms of BM, perhaps surprisingly the mice in the current study shifted from DR and back to AL feeding (DR-AL) remained lighter that AL mice at 1 month and 6 months post switch, only reaching a similar BM to AL mice 10 months after the dietary switch. In addition, those animals switched to DR in mid life (AL-DR), were still heavier than the early-life DR group following 1 month post switch, although by the 6-month timepoint their BM was indistinguishable from the DR group. While it has been reported that a shift back to AL feeding from acute DR (≤ 100 day duration) can increase BM back to AL levels within 50 days in MF1 mice , we observed a far slower response in agreement with studies in rats  and in other strains of mice . While the shift from DR-AL feeding induces hyperphagia, this response is suggested as being highly transient (1 to 5 days), after which food intake (FI) levels (and BM) return to AL levels [30, 31]. However, in rats the mass-adjusted FI of animals switched from DR-AL was elevated until the switch animals reached the similar BM of AL rats, around 18 months post switch . In our study, food intake in the DR-AL group was significantly greater at the 1-month and 6-month timepoints relative to AL mice. This hyperphagic response was not observed in the DR-AL group at the final timepoint, at a time when no BM differences were observed between the DR-AL and AL mice. Our data, as does the rat data , suggests that hyperphagia following DR is maintained until BM reaches the levels of AL animals. A similar prolonged hyperphagic response was also reported in male C57BL/6 mice following a dietary shift from DR-AL, although interestingly this was not observed in female mice . Significant differences in body composition (percentage lean and fat mass) were observed between groups throughout the timecourse of this experiment. At the 1-month timepoint, percentage lean mass was lowest, but percentage fat mass highest, in the AL-DR mice. This suggests that this group preferentially maintained their fat mass relative to lean mass following the mid-life switch to DR. The reasons for this are unclear, although may be a temporal stress response to an unpredictable food resource, as previously suggested . Interestingly, this response after 1 month of 30% DR may be age specific, as 3 weeks of 30% DR had no effect on either lean or fat mass in young (15 week old) male C57BL/6 (and DBA/2) mice , although it should be noted that this paper used a different method to control for BM differences between groups than used in the current manuscript. It was only after 10 months post switch that the DR-AL and AL-DR groups mirrored the body composition profiles of the early-life AL and DR groups, respectively.
In agreement with previous studies examining short-term DR in young mice [10–12], and in contrast to the body mass and body composition effects observed, mice switched from AL-DR rapidly adopted the improved glucose tolerance of the DR group. This similarity in glucose tolerance to mice exposed to DR from early life, and significant improvement relative to the AL mice, was maintained at both 6 months and 10 months post switch. These data indicate that DR, even when initiated in mid life, can induce significant, and rapid, benefits to the glycemic profile of mice, and support previous data in both rodents  and humans [13, 14, 34]. In mice switched from DR-AL, glucose tolerance was not statistically different to AL mice at 1 month post switch. However, by 6 months and 10 months post switch this group had a significantly enhanced glucose tolerance relative to the AL mice, despite having very similar body mass, body composition and fasting insulin levels to AL mice at the 10-month timepoint. These data suggest that male C57BL/6 mice retain a glycemic memory to previous DR feeding that maintains a long-term benefit to glucose tolerance that is independent of body composition and fasting insulin levels. In support, Cameron et al. also reported that male C57BL/6 mice switched from AL to DR (12 months of age) and then back to AL (17 months of age) had improved glucose tolerance relative to lifelong AL mice 5 months after switching back to AL feeding. The effect of dietary switching on fasting insulin levels and insulin sensitivity were less clear in our study, although the AL-DR had significantly lower fasting insulin levels and increased insulin sensitivity relative to both the DR-AL and AL mice 10 months post switch. Improved glucose tolerance following late-life initiation in DR has previously been reported in rats , although significant differences in serum insulin levels were only seen following glucose challenge . Fasting IGF-1 levels, which we have previously shown respond rapidly to early-life DR in mice , were also decreased significantly following mid-life DR, with the AL-DR group having lower IGF-1 levels compared to all other groups. This suggests that dietary switches to DR both in early life  and in mid life can elicit similar rapid changes in IGF-1 levels in mice, and that IGF-1 appears more responsive than insulin to DR . After 10 months post switch, while fasting insulin and IGF-1 levels were reduced and insulin sensitivity increased in the AL-DR group relative to AL mice, only IGF-1 levels were reduced in the DR-AL group compared to the AL group. The exact reasons for this uncoupling in the response of insulin and IGF-1 to mid-life dietary shifts back to AL feeding are currently unclear. We suggest that the precise role of insulin and IGF-1 signaling following mid-life dietary switches now requires further investigation, as late-onset DR in rats improved glucose tolerance without having any impact on insulin signaling within skeletal muscle . In addition, while many studies have examined the transcriptional response to DR in many tissues (see for example [15, 16, 18, 35, 36]), information on exactly how DR impacts on pancreatic and β-cell function is currently lacking. We suggest that with our data demonstrating clear evidence of a glycemic memory to previous DR exposure in mice, it is now critical to examine the molecular responses in the pancreas to DR and to mid-life dietary switches.