Research ArticleMICROBIOTA

Increasing breast milk betaine modulates Akkermansia abundance in mammalian neonates and improves long-term metabolic health

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Science Translational Medicine  31 Mar 2021:
Vol. 13, Issue 587, eabb0322
DOI: 10.1126/scitranslmed.abb0322
  • Fig. 1 Maternal betaine supplementation decreases adiposity in mouse offspring.

    (A) Shown are betaine relative concentrations in breast milk from control (blue) and betaine-treated (red) dams (n = 8 per group). (B and C) Shown is dam milk macronutrient composition (B) and energy content (C) (n = 5 per group). (D) Shown is plasma relative concentration of betaine in 2-week-old mice born to control dams (blue) or dams administered betaine (red) (n = 6 per group). (E) Shown is offspring weight gain during lactation (n = 36 per group, means ± SD). (F) Shown is milk intake at 1 week of age (pups grouped in litters, with n = 4 litters per group). (G) Body composition measured by MRI of 6-week-old offspring of control dams (n = 7, blue) or betaine-treated dams (n = 9, red) is shown. (H) mRNA expression of vWAT proinflammatory markers in 6-week-old male mice (n = 8 to 10 per group) is presented. (I) Plasma concentrations of proinflammatory markers Ccl2, IL-6, and Pai1 in 6-week-old male mice (n = 10 per group) are presented. (J) Shown is weight gain (means ± SD) during each week of lactation of offspring from obese control dams (MO-C, purple, n = 19) or betaine-treated obese dams (MO-B, orange, n = 21). (K) Shown is milk intake at 1 week of age for pups born to obese control dams (MO-C, purple) or betaine-treated obese dams (MO-B, orange) (pups grouped in litters, with n = 3 to 4 litters per group). (L) Shown is body composition in 6-week-old mice born to obese control dams (MO-C, purple) or betaine-treated obese dams (MO-B, orange) (n = 8 per group). Unless otherwise stated, data are means ± SEM. *, t test P < 0.05; **, t test P < 0.01; ***, P < 0.005; #, Mann-Whitney U test P < 0.05.

  • Fig. 2 Maternal betaine supplementation improves mouse offspring long-term metabolic health.

    (A to F) Offspring from control (blue) or betaine-treated (red) dams were fed a chow diet after weaning (n = 16 per group) and multiple parameters were measured. (A) Body weight throughout adulthood is shown. (B) Weight of liver, visceral WAT (vWAT), and subcutaneous WAT (scWAT) at euthanasia (24 weeks of age) are shown. (C and D) Shown is a glucose tolerance test (C) and insulin concentrations at 0 and 15 min after glucose load (D) in 20-week-old mouse offspring. (E) Shown is vWAT adipocyte area (scale bars, 100 μm), distribution, and average cell area in 24-week-old mice (n = 8 per group). (F) mRNA expression of proinflammatory markers in vWAT from control (blue) and betaine-treated (red) 24-week-old mouse offspring (n = 15 to 16 per group) is shown. (G to J) Offspring from obese control dams (MO-C, n = 11, purple) and betaine-treated obese dams (MO-B, n = 13, orange) were fed a chow diet after weaning and then multiple parameters were measured. (G) Body weight was monitored until adulthood. (H) Liver, vWAT, and scWAT weights at euthanasia (24 weeks of age) were measured. (I) Glucose tolerance and (J) insulin concentrations at 0 and 15 min after glucose load were measured in 20-week-old offspring. AUC, area under the curve. Data are means ± SEM. *, t test P < 0.05; **, P < 0.01; ***, P < 0.005.

  • Fig. 3 Maternal betaine supplementation increases Akkermansia abundance in neonatal mice.

    (A) PCoA of unweighted UniFrac distances for the cecal microbiota from 2- and 6-week-old offspring of betaine-treated dams (red) or control dams (blue) is presented; P value was assessed by the Adonis test (n = 10 per group). (B) Relative abundance of Akkermansia spp. in cecal samples from 2-week-old mice is shown as log(x + 1) values. (C) A. muciniphila copy number per mg of fecal sample in 1-month old (n = 19) and 12-month-old (n = 40) human infants is shown. Infants were categorized into low-exposure (blue triangles) and high-exposure (red triangles) groups based on milk betaine concentration median values (4.1 μM). (D) A. muciniphila was grown in vitro in the absence (blue) or presence (red) of 2.5 or 10 mM betaine until reaching the stationary phase (25 hours of growth); cell number was quantified by qPCR (n = 3, means ± SD; one-way ANOVA with post hoc Dunnett’s test). (E) Shown is goblet cell number in ileum sections from 2-week-old (n = 10 per group), 6-week-old (n = 16 per group), and 24-week-old (n = 6 to 7 per group) offspring born to control (blue) and betaine-treated (red) dams. (F to H) Shown are ileal mRNA concentrations of four intestinal barrier markers Muc2, Ocln, Zo1, and Zo2 in mouse offspring at (F) 2 weeks (n = 10 per group), (G) 6 weeks (n = 8 per group), and (H) 24 weeks of age (n = 8 to 10 per group). Unless otherwise stated, data are means ± SEM. *, Student’s t test P < 0.05; **, P < 0.01; #, Mann-Whitney U test P < 0.05.

  • Fig. 4 Early-life A. muciniphila exposure improves long-term metabolic health in mice.

    (A) Eleven-day-old male mice born to obese dams were administered PBS as control (MO-C, n = 16, blue) or pAkk (MO-Akk, n = 16, red) thrice weekly until day 21 and were then euthanized at 6 or 20 weeks of age (n = 8 per group for each time point). (B) Shown is weight gain for mouse offspring through days 11 to 21 (n = 16 per group). (C and D) Shown is vWAT and scWAT weight (C) and vWAT mRNA expression (D) in mouse offspring at 6 weeks of age (n = 8 per group). (E) Goblet cell number and (F) mRNA expression of intestinal barrier markers Muc2, Ocln, Zo1, and Zo2 in ileum of mouse offspring at 6 weeks of age (n = 8 per group) are shown. (G) Body weight was monitored until adulthood (n = 8 per group). (H) vWAT and scWAT weight in mice at 20 weeks of age is shown (n = 7 to 8 per group). (I) Glucose tolerance and (J) insulin concentrations at 0 and 15 min after glucose load in mice at 18 weeks of age (n = 8 per group) are shown. (K) Goblet cell number and (L) mRNA expression of intestinal barrier markers Muc2, Ocln, Zo1, and Zo2 in ileum of mice at 20 weeks of age (n = 7 to 8 per group) are shown. AUC, area under the curve. Data are means ± SEM. *, t test P < 0.05; **, P < 0.01; ***, P < 0.005.

  • Table 1 Demographic and metabolic characteristics of mother-infant dyads from cohorts I and II.

    Values represent means and SD.

    Cohort ICohort II
    Mothern = 34n = 109
    Age (years)29.1 ± 5.132.8 ± 6.2
    Prepregnancy BMI
    (kg/m2)
    27.3 ± 7.123.5 ± 3.8
    Gestational weight
    gain (kg)*
    12.0 ± 7.412.1 ± 4.7
    Infant
    Birthn = 34n = 109
    Gender (female/male)15/1960/49
    Gestational age
    (weeks)
    39.6 ± 1.239.6 ± 1.1
    Weight (kg)3.53 ± 0.483.35 ± 0.44
    Length (cm)51.6 ± 2.350.1 ± 2.2
    Weight-for-length
    z score
    −0.59 ± 1.2−0.20 ± 1.15
    1 monthn = 34n = 109
    Weight (kg)4.67 ± 0.714.23 ± 0.57
    Length (cm)55.8 ± 2.154.5 ± 2.3
    Weight-for-length
    z score
    −0.36 ± 1.04−0.50 ± 1.17
    12 monthsn = 107
    Weight (kg)9.46 ± 1.04
    Length (cm)74.8 ± 2.9
    Weight-for-length
    z score
    0.18 ± 0.87
    Breast milk
    metabolites
    n = 34n = 109
    SAM (nM)1469 ± 4362034 ± 649
    SAH (nM)216 ± 81327 ± 184
    Methionine (μM)4.39 ± 2.696.74 ± 12.36
    Cystathionine (nM)44.9 ± 34.3129.6 ± 75.7
    Betaine (μM)3.12 ± 2.655.33 ± 6.13
    Choline (μM)97 ± 49165 ± 115

    *n = 32 for gestational weight gain data in cohort I.

    • Table 2 Multivariate regression between human breast milk metabolite concentrations and infant growth.

      Least-square regression models were applied to assess the correlation between milk metabolite concentrations and change in weight-for-length z score from birth to 1 month or 12 months of age. B, size effect estimate; CI, confidence interval; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; Met, methionine; Cysta, cystathionine; Bet, betaine; Cho, choline. Bold font indicates P < 0.05.

      Cohort ICohort II
      Weight-for-length z score 1 monthWeight-for-length z score 1 monthWeight-for-length z score 12 months
      Adjusted*AdjustedAdjusted*AdjustedAdjusted*Adjusted
      n = 34n = 32n = 109n = 109n = 107n = 107
      B (CI 95%)PB (CI 95%)PB (CI 95%)PB (CI 95%)PB (CI 95%)PB (CI 95%)P
      SAM0.09
      (−1.08 to 1.25)
      0.879−0.12
      (−1.35 to 1.11)
      0.8410.52
      (−0.03 to 1.07)
      0.0660.55
      (0.01 to 1.10)
      0.0470.07
      (−0.39 to 0.53)
      0.7650.01
      (−0.45 to 0.48)
      0.964
      SAH−0.95
      (−1.91 to 0.01)
      0.053−0.88
      (−1.86 to 0.10)
      0.0770.10
      (−0.20 to 0.39)
      0.5250.12
      (−0.17 to 0.42)
      0.407−0.02
      (−0.26 to 0.22)
      0.848−0.06
      (−0.31 to 0.18)
      0.609
      Met0.10
      (−0.59 to 0.79)
      0.7720.39
      (−0.36 to 1.14)
      0.298−0.57
      (−0.87 to −0.26)
      0.001−0.51
      (−0.81 to −0.21)
      0.001−0.22
      (−0.47 to 0.04)
      0.094−0.18
      (−0.44 to 0.08)
      0.182
      Cysta−0.33
      (−0.68 to 0.02)
      0.061−0.35
      (−0.71 to 0.02)
      0.061−0.18
      (−0.57 to 0.2)
      0.341−0.22
      (−0.59 to 0.16)
      0.253−0.18
      (−0.48 to 0.13)
      0.258−0.17
      (−0.48 to 0.14)
      0.266
      Bet−0.66
      (−1.18 to −0.14)
      0.015−0.60
      (−1.14 to −0.07)
      0.028−0.56
      (−0.91 to −0.21)
      0.002−0.47
      (−0.83 to −0.11)
      0.010−0.38
      (−0.67 to −0.1)
      0.009−0.34
      (−0.64 to −0.04)
      0.026
      Cho−0.23
      (−0.92 to 0.46)
      0.503−0.26
      (−1.03 to 0.51)
      0.497−0.28
      (−0.67 to 0.10)
      0.148−0.23
      (−0.61 to 0.15)
      0.237−0.21
      (−0.53 to 0.11)
      0.190−0.17
      (−0.49 to 0.15)
      0.287

      *Adjusted for gestational age and weight-for-length z score at birth.

      †Adjusted for gestational age, weight-for-length z score at birth, prepregnancy BMI, gestational weight gain, and mode of birth.

      Supplementary Materials

      • stm.sciencemag.org/cgi/content/full/13/587/eabb0322/DC1

        Fig. S1. Effect of maternal betaine administration on young mouse offspring.

        Fig. S2. Effects of maternal betaine administration on mouse offspring energy homeostasis.

        Fig. S3. Effects of maternal antibiotic coadministration on offspring long-term metabolic health in mice.

        Fig. S4. Effect of betaine administration on the maternal and offspring gut microbiome in mice.

        Fig. S5. Effect of betaine on bacterial growth in vitro.

        Fig. S6. Effect of maternal betaine supplementation on mouse ileum histology and gene expression.

        Table S1. No association between milk betaine concentration and change in human infant body length z score and head circumference.

        Table S2. Prevalence of A. muciniphila in human infants exposed to low and high breast milk betaine content.

        Table S3. Primer sequences for qPCR analyses.

        Data file S1. Individual level data for all figures.

      • The PDF file includes:

        • Fig. S1. Effect of maternal betaine administration on young mouse offspring.
        • Fig. S2. Effects of maternal betaine administration on mouse offspring energy homeostasis.
        • Fig. S3. Effects of maternal antibiotic coadministration on offspring long-term metabolic health in mice.
        • Fig. S4. Effect of betaine administration on the maternal and offspring gut microbiome in mice.
        • Fig. S5. Effect of betaine on bacterial growth in vitro.
        • Fig. S6. Effect of maternal betaine supplementation on mouse ileum histology and gene expression.
        • Table S1. No association between milk betaine concentration and change in human infant body length z score and head circumference.
        • Table S2. Prevalence of A. muciniphila in human infants exposed to low and high breast milk betaine content.
        • Table S3. Primer sequences for qPCR analyses.

        [Download PDF]

        Other Supplementary Material for this manuscript includes the following:

        • Data file S1 (Microsoft Excel format). Individual level data for all figures.

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