Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice

Abstract

Dietary restriction (DR) extends lifespan and increases resistance to multiple forms of stress, including ischemia reperfusion injury to the brain and heart in rodents. While maximal effects on lifespan require long-term restriction, the kinetics of onset of benefits against acute stress is not known. Here, we show that 2-4 weeks of 30% DR improved survival and kidney function following renal ischemia reperfusion injury in mice. Brief periods of water-only fasting were similarly effective at protecting against ischemic damage. Significant protection occurred within 1 day, persisted for several days beyond the fasting period and extended to another organ, the liver. Protection by both short-term DR and fasting correlated with improved insulin sensitivity, increased expression of markers of antioxidant defense and reduced expression of markers of inflammation and insulin/insulin-like growth factor-1 signaling. Unbiased transcriptional profiling of kidneys from mice subject to short-term DR or fasting revealed a significant enrichment of signature genes of long-term DR. These data demonstrate that brief periods of reduced food intake, including short-term daily restriction and fasting, can increase resistance to ischemia reperfusion injury in rodents and suggest a rapid onset of benefits of DR in mammals.

Figures

Figure 1. DR protects against the damaging effects of renal IRI

A. Survival curves of male mice fed ad libitum, restricted to 70% of ad libitum food consumption for 4 weeks, or fasted for 3 days prior to induction of 37 minutes of bilateral renal IRI (n=10 per group). No further mortality was observed beyond day 4 after surgery. Both dietary treatments led to a significant survival advantage by Kaplan Meier analysis (log rank test, p

Figure 2. Rapid onset and loss of protective effects of short-term DR and fasting

A-C. Rapid onset: A. Survival curves of male mice fed ad libitum, 30% restricted for 2–4 weeks, or fasted for 1–3 days prior to induction of 37 minutes of unilateral renal IRI with contralateral nephrectomy (n=10–18 per group). B. Kidney function as measured by serum urea following 37 minutes of unilateral renal IRI in the indicated groups (serum from 4–10 individual animals was sampled per data point). Asterisks indicate the significance of the difference as compared to the ad libitum group at the same time point (** p99mTc-DMSA, expressed as a percentage of the injected dose per one kidney (% ID/kidney). Note the reduced percentage of 99mTc-DMSA in the kidneys of the ad libitum group 24 hours after renal IRI (12.4% to 5.6%), indicative of renal dysfunction. The significance of the difference as compared to the ad libitum group prior to (asterisks) or one day after (crosses) renal IRI is indicated. D, E. Rapid loss: D. Survival curves of the indicated groups. Survival of animals refed for 2hr, 1 and 2 days was significantly different than ad libitum fed animals (p<0.002); survival of animals refed for 4 and 7 days was not significantly different than ad libitum fed animals. E. Kidney function as measured by serum urea prior to and one day following IRI. Data from three independent experiments with 4–12 animals per group are averaged. In the day 0 group asterisks indicate significant differences vs. the ad libitum fed control group; in the day 1 group, asterisks indicate significant difference between 3 days of fasting without refeeding and ad libitum (AL) fed animals as well as each of the refed groups (**p<0.01). There were no significant differences between the ad libitum group and any of the refed groups on day 1 following renal IRI.

Figure 3. Protection is a preconditioning effect

A. Black bars indicate periods of free access to chow; grey bars indicate periods without access to chow. Body weights of the two groups at the time of surgery (day 0) are indicated. Error bars indicating SEM are contained within the symbols. B. Survival following renal IRI; fasted animals retained their survival advantage (p

Figure 4. Dietary preconditioning in the liver

A. Reduced injury markers upon liver IRI in fasted mice. Mice (5–8 animals per group) were fasted for the indicated times prior to induction of 75 minutes of warm ischemia to the liver. Serum concentration of the liver-specific enzyme alanine aminotransferase (ALAT) indicative of liver damage was measured at the indicated times following reperfusion. Asterisks indicate the significance of the difference as compared to the ad libitum group at the same time point using a Mann-Whitney U-test (* p

Figure 5. Insulin and IGF-1 signaling in dietary preconditioning

A. Improved insulin sensitivity in preconditioned mice. Whole blood glucose levels at the indicated timepoints following intraperitoneal injection of insulin into animals following the indicated preconditioning regimens. Right: area under the curves (AUC). Statistically significant differences relative to the ad libitum group are indicated by asterisks (** p

Figure 6. Global transcriptional changes upon short-term dietary restriction and fasting

A. Venn diagram representing numbers and overlap of probesets significantly differentially regulated in kidney as a result of dietary preconditioning (4 wk 30% DR or 3 days fasting, FA) as compared to ad libitum fed controls. Significance cutoffs were set by fold change of greater than 1.5 and p value of 1.5 and p value