Figure 1. Isoflurane exposure disrupts circulating glucose and beta-hydroxybutyrate in neonatal mice.

(A) Blood β-hydroxybutyrate (β-HB) concentration in neonatal post-natal day 7 (P7), adolescent post-natal day 30 (P30), and young adult post-natal day 60 (P60) mice. n = 17, 19, and 9, respectively. ****p<0.0001 by pairwise t-test. ANOVA ****p<0.0001. (B) Blood glucose in P7, P30, and P60 mice. n = 14, 13, and 9, respectively. ****p<0.0001 by pairwise t-test. ANOVA ****p<0.0001. (C, D) Blood β-HB in P7 neonatal mice exposed to 1.5% isoflurane or control conditions for 0 to 120 min. (C) Pairwise comparisons shown between baseline (time = 0) and respective treatment timepoints. *p≤0.05, ****p<0.0001 by two-tailed pairwise t-test. (D) Bar graphs with individual datapoints for pairwise comparisons between treatments at 30, 60, and 120 min. ****p<0.0001 by two-tailed pairwise t-test. (E, F) Blood β-HB in P30 adolescent mice exposed to 1.5% isoflurane or control conditions for 0–120 min. (E) Control and isoflurane-exposed groups both show a modest but significant increase in β-HB over baseline by 2 hr of exposure, *p≤0.05. (F) Isoflurane exposure did not significantly alter β-HB levels relative to time-matched control conditions. (G–H) Blood β-HB in P7 neonatal mice exposed to 1.5% isoflurane or control conditions for 0–120 min. (G) Pairwise comparisons shown between baseline (time = 0) and respective treatment timepoints. *p≤0.05, **p<0.005, ***p<0.0005, ****p<0.0001 by two-tailed pairwise t-test. (H) Bar graphs with individual datapoints for pairwise comparisons between treatments at 30, 60, and 120 min. *p≤0.05, **p<0.005. (I, J) Blood glucose levels in P30 mice exposed to control conditions or 1.5% isoflurane anesthesia for up to 120 min. (I) Blood glucose levels did not significantly change compared to baseline. (J) Bar graphs of data in (I) with individual datapoints for pairwise comparison of blood glucose levels by treatment. No significant changes observed. (K, L) Blood glucose in P7 mice provided glucose by IP injection at the start of anesthetic exposure plotted as a function of time. (K) Control-exposed and 1.5% isoflurane- exposed data from (G) shown for reference. Pairwise comparisons shown between baseline (T = 0) values and 15, 30, and 60 min timepoints in 1.5% isoflurane (+) glucose treatment group, ****p<0.0001 by pairwise t-test. (L) Bar graphs of (K) with individual datapoints for pairwise comparisons of blood glucose in mice exposed to 1.5% isoflurane or 1.5% isoflurane (+) glucose. ****p<0.0001 by two-tailed pairwise t-test. (M, N) Blood β-HB levels in mice provided glucose by IP injection at the start of anesthetic exposure, plotted as a function of time. (M) Control-exposed and 1.5% isoflurane-exposed data from (C) shown for reference. Pairwise comparisons shown between baseline (T = 0) and 15, 30, and 60 min timepoints in 1.5% isoflurane (+) glucose treatment group, ****p<0.0001. (N) Bar graphs of (M) with individual datapoints for pairwise comparisons of blood β-HB in mice exposed to 1.5% isoflurane or 1.5% isoflurane (+) glucose. Glucose administration did not attenuate the loss of β-HB in response to isoflurane exposure. (O, P) Blood β-HB levels in mice provided β-HB by IP injection at the start of anesthetic exposure. (O) Control-exposed and 1.5% isoflurane-exposed data from (C) shown for reference. Pairwise comparisons shown between baseline (T = 0) and 60 and 120 min timepoints in 1.5% isoflurane (+) β-HB treatment group, ****p<0.0001. (P) Bar graphs of (O) with individual datapoints for pairwise comparisons of blood β-HB in mice exposed to 1.5% isoflurane or 1.5% isoflurane (+) glucose. ****p<0.0001. (Q, R) Blood glucose levels in mice provided β-HB by IP injection at the start of anesthetic exposure, plotted as a function of time. (Q) Control -exposed and 1.5% isoflurane-exposed data from (G) shown for reference. Pairwise comparisons shown between baseline (T = 0) and 60 and 120 min timepoints in 1.5% isoflurane (+) β-HB treatment group, ****p<0.0001, n.s. – not significant. (R) Bar graphs of (Q) with individual datapoints for pairwise comparisons of blood β-HB in mice exposed to 1.5% isoflurane or 1.5% isoflurane (+) glucose or baseline. *p<0.05. For all data, error bars represent standard error of the mean (SEM). ANOVA p-value for 1 hr dataset *p=0.0083; ANOVA for 2 hr dataset **p=0.0033. (A–R) For all data, n ≥ 3 per time/treatment. Each datapoint in bar graphs represents an individual animal. See Materials and methods for additional details.

Figure 1—figure supplement 1. Dose-dependent effects of 1% and 1.5% isoflurane on blood lactate and glucose in P7 mice.

(A) Blood lactate as a function of time in mice exposed to control conditions in either air or 100% oxygen for up to 2 hr. (B) Bar graphs of 60 min data from (A). One-way ANOVA *p<0.05. *p<0.05 by pairwise t-test. (C) Bar graphs of 120 min data from (A). (D) Whole-blood β-HB in P7 neonatal mice exposed to 1.5%, 1% isoflurane, or control conditions for 0 to 120 min. (A, D) *p≤0.05, **p<0.005, ***p<0.0005, ****p<0.0001 by two-tailed pairwise t-test versus baseline (time = 0). (B, C) One-way ANOVA ***p=0.0001, *p<0.05 and ***p<0.0005 by pairwise t-test.

Figure 1—figure supplement 2. Circulating nutrient factors.

Brief exposure to isoflurane results in significant changes to glucoregulatory factors, but directionality is inconsistent with a role in driving the metabolic effects reported here. (A) Insulin, the major circulating factor responsible for glucose uptake by tissue; (B) c-peptide, a peptide released as a byproduct of insulin production; (C) gastric inhibitory polypeptide (GIP), a factor involved in stimulating insulin secretion; (D) glucagon, a key hypoglycemia response factor which drives glucose release and gluconeogenesis; (E) pancreatic polypeptide (PP), a pancreatic secretion regulator induced by acute hypoglycemia or fasting and decreased by high levels of glucose. *p≤0.05, **p≤0.005 by two-tailed pairwise t-test.

Figure 1—figure supplement 3. Oxygen concentration does not impact circulating glucose or β-HB.

(A) Blood β-HB as a function of time in P7 mice exposed to control conditions in either air or 100% oxygen for up to 2 hr. No significant differences were observed at any timepoint. (B) Blood glucose as a function of time in mice exposed to control conditions in either air or 100% oxygen for up to 2 hr. No significant differences were observed at any timepoint. N ≥ 3 at each timepoint.