Exercise-induced 3′-sialyllactose in breast milk is a critical mediator to improve metabolic health and cardiac function in mouse offspring

Abstract

Poor maternal environments, such as under- or overnutrition, can increase the risk for the development of obesity, type 2 diabetes and cardiovascular disease in offspring1,2,3,4,5,6,7,8,9. Recent studies in animal models have shown that maternal exercise before and during pregnancy abolishes the age-related development of impaired glucose metabolism10,11,12,13,14,15, decreased cardiovascular function16 and increased adiposity11,15; however, the underlying mechanisms for maternal exercise to improve offspring’s health have not been identified. In the present study, we identify an exercise-induced increase in the oligosaccharide 3′-sialyllactose (3′-SL) in milk in humans and mice, and show that the beneficial effects of maternal exercise on mouse offspring’s metabolic health and cardiac function are mediated by 3′-SL. In global 3′-SL knockout mice (3-SL−/−), maternal exercise training failed to improve offspring metabolic health or cardiac function in mice. There was no beneficial effect of maternal exercise on wild-type offspring who consumed milk from exercise-trained 3-SL−/− dams, whereas supplementing 3′-SL during lactation to wild-type mice improved metabolic health and cardiac function in offspring during adulthood. Importantly, supplementation of 3′-SL negated the detrimental effects of a high-fat diet on body composition and metabolism. The present study reveals a critical role for the oligosaccharide 3′-SL in milk to mediate the effects of maternal exercise on offspring’s health. 3′-SL supplementation is a potential therapeutic approach to combat the development of obesity, type 2 diabetes and cardiovascular disease.

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Fig. 1: Exercise-induced adaptations to milk improve metabolic health and cardiac function in offspring.
Fig. 2: Maternal exercise increases 3′-SL in milk in humans and rodents, and 3′-SL is required for the beneficial effects of maternal exercise to mediate improvements in offspring’s metabolic health and cardiac function.
Fig. 3: Milk from exercise-trained, 3-SL/ dams did not improve metabolic health or cardiac function of WT mice.
Fig. 4: Supplementation of 3′-SL during the lactation period improves metabolic health and cardiac function of adult offspring.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH grants (nos. R01HL138738 to K.I.S. and R01AG060542 to K.I.S. and M.T.Z., and R01-DK101043 to L.J.G.), and the Joslin Diabetes Center DRC (grant no. P30 DK36836). K.M.P. was supported by T32HL134616. The authors thank P. J. Mohler and E. D. Lewandowski for critical discussions.

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Contributions

J.E.H, K.M.P. and K.R.W. performed exercise-training experiments, supplementation of 3′-SL, metabolic and cardiac experiments with mice, and measurements of gene expression. L.A.B., P.J.A. and A.C.L. performed exercise-training experiments, supplementation of 3′-SL and metabolic experiments with mice. K.M. and T.J.C. performed measurements of gene expression. E.A. and V.K.S. performed in vivo cardiac measurements. B.R. performed HPLC in breast milk. L.J.G. supervised in vivo experiments in mice and assisted in writing the manuscript. C.S. and A.A. supervised the clinical study. M.T.Z. supervised and analysed cardiac data and assisted in writing the manuscript. L.B. supervised experiments involving breast milk and HPLC. K.I.S. directed the research project, designed and carried out experiments, analysed data and wrote the manuscript.

Corresponding author

Correspondence to Kristin I. Stanford.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Effects of maternal exercise in dams.

6’SL in human participants (a) average activity during pregnancy, (b) average steps per day during pregnancy, and (c) BMI; N=139, data analyzed by Pearson’s correlation. (d) 6’SL in breastmilk from sedentary and exercise-trained chow-fed and high-fat fed dams. Data are expressed as the mean ± SEM (Sed Chow n=7; Train Chow n=7; Sed High-Fat n=6; Train High-Fat n=8). There was no difference in (e) citrate synthase of (f) hexokinase 2 (HK2) protein expression in tibialis anterior skeletal muscles of wild-type (WT) and 3’SL−/− exercise-trained dams (n=3/group). (g) ITT, and (g) ejection fraction in male offspring from sedentary or exercise-trained wild-type or 3’SL−/− dams, and (i) ITT in female offspring from sedentary or exercise-trained wild-type or 3’SL−/− dams. Data are expressed as the mean ± SEM (for males, WT Sed n=18, WT Train n=21, 3’SL−/− Sed n=9, 3’SL−/− Train n=9. For females, WT Sed n=24; WT Train n=22; 3’SL−/− Sed n=5; 3’SL−/− Train n=10). (j) Exercise capacity test in wild-type or 3’SL−/− male mice (n=6/group). Asterisks represent differences compared to WT (***P < 0.001). (k) GTT by lean mass and wild-type or 3’SL−/− male or female mice at 24 wks of age. Data are expressed as the mean ± SEM (n=6 WT males; n=7 WT females; n=15; 3’SL−/− males; n=8; 3’SL−/− females). Asterisks represent differences compared to WT of same gender (*P < 0.05). (l) Body weight; (m) fat mass; (n) lean mass; (o) glucose excursion curve; and (p) GTT AUC in 12-week-old male WT (n=13) or 3’SL-/- (n=5) mice placed on a high-fat diet for 6 weeks. Data are expressed as the mean ± SEM. Asterisks represent differences compared to 3’SL-/- (*P < 0.05; **P<0.01). (q) Body weight, (r) fat mass, (s) glucose tolerance excursion curve, and (t) GTT AUC in female offspring from wild-type sedentary (n=22) or exercise-trained dams (n=22), 3’SL-/- sedentary dams (n=12), or 3’SL-/- sedentary dams cross-fostered to sedentary wild-type dams (3’SL-WT) (n=6). Data are expressed as the mean ± SEM. Asterisks represent differences compared to WT Sedentary offspring (*P < 0.05; **P < 0.01; ***P < 0.001); and # represents differences compared to WT Trained offspring (###P<0.01). Two-way ANOVA was used for g,i,o, and s with Tukey’s multiple comparisons tests; one-way ANOVA was used for d,h,q,r, and t, with Tukey’s multiple comparisons tests; unpaired two-tailed Student’s t-test was used for e,f,j,k,l,m,n, and p. Source data

Extended Data Fig. 2 Maternal exercise alters expression of hepatic genes.

Expression of hepatic genes involved in (a,b) gluconeogenesis and glucose metabolism, (c,d) mitochondrial activity and Krebs Cycle activity, and (e,f) fatty acid transport and oxidation and male and female offspring from wild-type (WT) and 3’SL-/- mice. Data are expressed as the mean ± SEM (for males, WT Sed n=8, WT Train n=5, 3’SL-/- Sed n=6, 3’SL-/- Train n=6. For females, WT Sed n=9, WT Train n=4, 3’SL-/- Sed n=6, 3’SL-/- Train n=8). Asterisks represent differences compared to WT Sedentary offspring (*P < 0.05; ***P<0.001) or compared to 3’SL-/- Sedentary offspring (#P<0.05). One-way ANOVA was used for a-f with Tukey’s multiple comparisons tests.

Extended Data Fig. 3 Maternal exercise alters expression of cardiac genes.

Expression of cardiac genes involved in (a,b) mitochondrial activity, (c,d) fibrosis, and (e,f) fetal reprogramming and male and female offspring from WT and 3’SL-/- mice. Data are expressed as the mean ± SEM (for males, WT Sed n=9, WT Train n=9, 3’SL-/- Sed n=6, 3’SL-/- Train n=5 for Figure a; WT Sed n=9, WT Train n=5, 3’SL-/- Sed n=6, 3’SL-/- Train n=5 for Figures c, e. For females, WT Sed n=9, WT Train n=10, 3’SL-/- Sed n=4, 3’SL-/- Train n=4 for Figures b, d; WT Sed n=9, WT Train n=4, 3’SL-/- Sed n=4, 3’SL-/- Train n=4 for Figure f). Asterisks represent differences compared to Sedentary offspring of the same gender (*P < 0.05; **P < 0.01) or compared to 3’SL-/- Sedentary offspring (#P<0.05). One-way ANOVA was used for a-f with Tukey’s multiple comparisons tests.

Extended Data Fig. 4 Supplementation with 3’SL improves metabolic health of offspring.

(a) Body weight, (b) % fat mass, (c) % lean mass, and (d) GTT AUC in male mice supplemented with 300 (n=7), 600 (n=26), 900 (n=5), or 1200 (n=5) nmol/day of 3’SL, or PBS fed (n=10). Data are expressed as the mean ± SEM. Asterisks represent differences compared to PBS fed (***P<0.001). (e) Body weight, (f) % fat mass, (g) % lean mass, and (h) GTT AUC in female mice supplemented with 300 (n=7), 600 (n=24), 900 (n=4), or 1200 (n=5) nmol/day of 3’SL, or PBS fed (n=18). Data are expressed as the mean ± SEM. Asterisks represent differences compared to PBS fed (**P<0.01). (i) Body weight, (j) % fat mass, (k) % lean mass, and (l) GTT AUC in male mice supplemented with 600 nmol/day 3’SL (n=26), 300 nmol/day 6’SL (n=5), or PBS fed (n=10). Data are expressed as the mean ± SEM. Asterisks represent differences compared to PBS fed (***P<0.001). (m) Body weight, (n) % fat mass, (o) % lean mass, and (p) GTT AUC in female mice supplemented with 600 nmol/day 3’SL (n=24), 300 nmol/day 6’SL (n=9), or PBS fed (n=18). Data are expressed as the mean ± SEM. (q) 3’SL in circulation of male offspring from sedentary dams (n=2), exercise-trained dams (n=3), or pups fed 3’SL (n=3). Data are expressed as the mean ± SEM. Asterisks represent differences compared to sedentary (*P<0.05). Two-way ANOVA was used for a,e,i, and m with Tukey’s multiple comparisons tests; one-way ANOVA was used for b,c,d,f,g,h,j,k,l,n,o,p and q with Tukey’s multiple comparisons tests.

Extended Data Fig. 5 Supplementation of 3’SL alters expression of hepatic and cardiac genes.

Expression of hepatic genes involved in (a) gluconeogenesis and glucose metabolism, (b) fatty acid transport and metabolism, (c) mitochondrial activity, and (d) Krebs Cycle activity in male and female from PBS and 3’SL fed offspring. Expression of cardiac genes involved in (e) fetal programming genes, (f) mitochondrial activity, and (g) fibrosis in male and female offspring from PBS and 3’SL fed offspring. Data are expressed as the mean ± SEM (for males, PBS Fed n=6, 3’SL Fed n=17. For females, PBS Fed n=7, 3’SL Fed n=7 for Figures a-d; PBS Fed n=15, 3’SL Fed n=11 for Figures e-g). Asterisks represent differences compared to PBS fed offspring of the same gender (*P < 0.05; **P < 0.01; ***P < 0.001). One-way ANOVA was used for a-g with Tukey’s multiple comparisons tests.

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Source Data Extended Data Fig. 1

Unprocessed western blots of citrate kinase and hexokinase II.

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Harris, J.E., Pinckard, K.M., Wright, K.R. et al. Exercise-induced 3′-sialyllactose in breast milk is a critical mediator to improve metabolic health and cardiac function in mouse offspring. Nat Metab (2020). https://doi.org/10.1038/s42255-020-0223-8

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