Some believe DR could benefit human health. While it is still not known whether DR enhances human longevity, DR appears to reduce blood pressure, cholesterol, and insulin resistance (Fontana 2008, Rae 2004). DR may also affect the brain (Duan & Mattson 1999, Yu & Mattson 1999), and it was recently reported that 30% CR improved memory test performance in a human cohort (Witte et al. 2009). Mechanistic-oriented studies also suggest DR affects brain physiology. CR-treated rats have increased brain mitochondrial DNA (mtDNA) levels (Cassano et al. 2006). In one study, compared to AL mice the brains of alternate day fed mice showed increased oxygen consumption and mitochondrial biogenesis (Nisoli et al. 2005). CR reduced brain amyloidosis in tg2576 mice that express a mutated human amyloid precursor protein gene (Wang et al. 2005).

The effects of DR on several proteins and pathways that sense cell nutrient and bioenergetic status were assessed in mouse brain protein lysates. To minimize the effects of post-mortem changes, the mice were anesthetized and sacrificed by microwave irradiation of the whole brain. For mice in the DR groups, protein lysates were prepared from mice at the end of a 24 hour fasting cycle.

We evaluated the status of the insulin signaling pathway. AKT is an insulin-signaling pathway serine/threonine kinase that plays a role in metabolic signaling, contributes to glucose homeostasis, and is activated via phosphorylation (Manning & Cantley 2007). A Western blot performed using an AKT-Ser473P specific primary antibody showed reduced AKT phosphorylation at serine 473 in both DR groups (Figure 3a). Reduced AKT-Ser473P was still evident after normalizing to total AKT (which was unchanged by DR) and to GAPDH (Figure 3b-d). A Western blot performed using an antibody specific for GSK3β phosphorylation at Ser9 also found reduced GSK3β phosphorylation at that site (Figure 4a). Reduced phosphorylation of GSK3β-Ser9 was still evident after normalizing to total GSK3β (which was unchanged by DR) and to GAPDH (Figure 4b-d).

We also used a Western blot immunochemistry approach to investigate the status of proteins that sense cell energy supplies, influence bioenergetic metabolism, and are influenced by cell bioenergetic demands. The AMPK enzyme is activated by an increase in the cytosolic AMP to ATP ratio (Hardie 2007). High ratios are associated with AMPK phosphorylation at Thr172 (Scharf et al. 2008), and this AMPK phosphorylation is often used as a surrogate measure of its activation status. Very little AMPK-Thr172P was seen in our brain homogenates, and between the groups there was no difference in AMPK-Thr172 (Figure 5a). AMPK-Thr172P levels were still equivalent after normalizing to total AMPK and GAPDH (Figure 5b-d). Consistent with the low level of AMPK-Thr172P observed, we measured brain ATP levels in perchloric acid-prepared brain homogenates and detected relatively high and quantitatively equivalent levels across groups (Figure 6). In other experiments in which AL and DR mice were sacrificed by decapitation, brain homogenates showed more robust, quantitatively equivalent AMPK-Thr172P staining and nearly undetectable ATP levels (data not shown).

SIRT1 is an NAD+-dependent histone deacetylase that is believed to mediate some of the benefits of DR, and also to upregulate aerobic metabolism (Guarente 2007, Nemoto et al. 2005). It is reported that SIRT1 phosphorylation as indicated by the phosphorylation-specific Ser47 SIRT1 antibody is a marker of SIRT1 activation (Sasaki et al. 2008). In our DR experiments SIRT1 phosphorylation was found to be equivalent between groups (Figure 7a). Total SIRT1 levels were also equivalent between the groups (Figure 7b). Normalizing SIRT1-P to either SIRT1 or GAPDH also did not reveal a DR effect (Figure 7c-d).

SIRT1 and AMPK reportedly activate PGC1a, a “master regulator” of mitochondrial mass and aerobic metabolism (Handschin & Spiegelman 2006, Finck & Kelly 2006, Houten & Auwerx 2004). This transcriptional co-activator promotes both its own expression and mitochondrial biogenesis (Mootha et al. 2004, Schreiber et al. 2003). Total PGC1a levels were equivalent between groups, which suggests our DR regimen did not induce PGC1a activation in the brains of young male C57BL/6 mice (Figure 8). Consistent with this, levels of COX4 protein, a nuclear encoded protein that constitutes part of cytochrome oxidase and which is often used as a marker of mitochondrial mass, were unchanged by DR (Figure 9).

Studies by others report DR profoundly affects liver metabolism. Liver effects include increased gluconeogenesis, mitochondrial proliferation, and oxygen consumption (Civitarese et al. 2007, Hagopian et al. 2003, Rodgers et al. 2005). Severe DR also results in liver ketone body production (Owen et al. 1969, Owen et al. 1967). These adaptive changes ensure the brain receives an uninterrupted supply of energy substrates, either in the form of glucose or as a combination of glucose and the ketone bodies betahydroxybutyrate and acetoacetate. In our study the greatest degree of nutrient deprivation occurred at the end of a 24 hour fast, at which point blood glucose levels were lower than they were in non-fasting mice. Although blood glucose levels declined with fasting, for mice repeatedly cycling through a 24 hour fast period this decline was maximal at 6 hours. During the entire 24 hour fast period frank hypoglycemia was avoided. Although we did not specifically test the mechanisms that stabilized glucose levels in our fasting mice, it seems likely this stability reflects the ability of liver gluconeogenesis to maintain a fairly constant blood glucose level over the course of a 24 hour fast.

Overall reductions in nutrient intake also affect insulin production by the pancreas. DR is associated with reduced insulin release and increased muscle, liver, and adipose insulin sensitivity (Kemnitz et al. 1994). Blood glucose levels were lower in our DR mice than in our control mice during periods where both groups had unlimited access to food, and also when both groups were subjected to a six hour fast. This suggests DR enhanced insulin sensitivity in the mice studied in these experiments. Also, aged C57BL/6 mice maintained on an otherwise identical IF diet showed enhanced recovery from a GTT glucose challenge, and aged IF and IF+AO mice showed greater ITT responses to an insulin challenge than aged AL mice. While it remains important to note we did not directly measure blood insulin levels or perform glucose and insulin tolerance tests in our young mice, literature data clearly indicate DR reduces insulin secretion (Merry 2002). It therefore seems reasonable to assume insulin levels in our DR mice were likely lower than they were in our AL mice. In future studies we would like to directly address this by measuring blood insulin levels.

Reduced insulin signaling in the brains of our DR mice could reflect either brain insulin resistance or reduced insulin in the brain. As there is no reason to expect DR would increase insulin resistance in any tissue the latter possibility seems more likely. Reduced pancreas insulin secretion leading to reduced plasma insulin should also predictably lower the brain insulin level.

While AKT phosphorylation is not solely determined by the insulin signaling pathway, we suspect reduced insulin signaling in DR-treated mouse brains accounts for the observed reduction in their AKT ser473 phosphorylation. We were also able to show phosphorylation of GSK3β, a target of the AKT kinase, was decreased. Reduced GSK3β phosphorylation is associated with increased GSK3β activity (Grimes & Jope 2001). In neurons, increased GSK3β activity promotes tau phosphorylation (Bhat & Budd 2002). A prior study of C57BL/6 mice found 24-72 hour fasts increased brain tau phosphorylation (Yanagisawa et al. 1999). Our finding that GSK3β phosphorylation is decreased, most likely as a consequence of reduced AKT phosphorylation, is consistent with the results of that study.

The 6 week DR protocol we used did not appear to induce brain mitochondrial biogenesis in young male C57BL/6 mice. Other studies, though, have reported DR potentially induces brain mitochondrial biogenesis. A study done in rats found mtDNA content was increased by a DR protocol (Cassano et al. 2006). Of more direct relevance is the study of Nisoli et al, who did report evidence of brain mitochondrial biogenesis in mice fed on alternate days (Nisoli et al. 2005). Specifically, increased brain PGC1a mRNA expression, increased SIRT1 protein, increased COX4 protein, and increased oxygen consumption were detected. We did not measure brain PGC1a transcript levels nor oxygen consumption. We did not find increased SIRT1 or COX4 protein, but a number of methodologic issues may account for differences between that and our study. The mice in the Nisoli et al study were strain hybrids, they were older than our mice, they were sacrificed at the end of a feeding cycle, and they were maintained on RFR diets for longer periods (3-12 months). It is possible our small sample sizes may have caused a type II error. It is also clear other technical issues can influence relevant measurements, as evidenced by the fact that we detected very low ATP levels in the brains of mice sacrificed by decapitation, and much higher ATP levels in the brains of mice sacrificed by microwave irradiation. Other investigators have also experienced this, and some report microwave irradiation likely permits a more accurate assessment of brain ATP levels (Scharf et al. 2008). In our study neither approach revealed inter-group differences in brain ATP levels. Regardless, differences between the models and protocols used by us and other investigators preclude gross generalizations as to whether DR can or cannot induce mitochondrial biogenesis.

Because of the study of Nisoli et al, which found RFR does induce brain mitochondrial biogenesis in mice (Nisoli et al. 2005), and the study of Schulz et al, which found antioxidants mitigate increased respiration in 2-deoxyglucose-treated C. elegans (Schulz et al. 2007), we included a DR group that was co-treated with an antioxidant cocktail. We were, however, unable to critically test whether antioxidant supplementation reduced brain mitochondrial biogenesis in our RFR mice because even without antioxidant supplements we did not detect evidence of brain mitochondrial biogenesis. It is worth commenting, though, on the nature of our antioxidant cocktail. Blueberry, pomegranate, and EGCG extracts reportedly constitute antioxidants that reduce morbidity (Seeram 2008, Khan & Mukhtar 2007). We wanted to avoid antioxidants that may induce morbidity, such as might be the case with high dose vitamin E supplementation (Bjelakovic et al. 2007, Miller et al. 2005). In retrospect, blueberry, pomegranate, and EGCG extracts may have actions that extend beyond their ability to reduce oxidative stress (Singh et al. 2008), and it could be argued more traditional antioxidants should have been selected.

While it is possible other brain-specific compensatory mechanisms may have occurred to maintain brain ATP levels during DR, the fact that frank hypoglycemia did not develop during the course of a 24 hour fast suggests the brains of our DR mice were at no point deprived of energy substrates. If this is correct, a need to increase brain ATP levels by upregulating aerobic metabolism may not have occurred. The ability of liver gluconeogenesis to generate adequate glucose to fuel the brain over a 24 hour period may have cushioned the direct brain effects of the DR, and mitigated any need to activate AMPK or SIRT1 within the brain. This could potentially explain why we observed neither an increase in the brain PGC1a level nor an increase in COX4 protein.