Am J Physiol Endocrinol Metab 306: E838–E847, 2014.
First published February 11, 2014; doi:10.1152/ajpendo.00605.2013.
De novo synthesis of milk triglycerides in humans
Mahmoud A. Mohammad, Agneta L. Sunehag, and Morey W. Haymond
Department of Pediatrics, Children’s Nutrition Research Center, US Department of Agriculture/Agricultural Research
Service, Baylor College of Medicine, Houston, Texas
Submitted 4 November 2013; accepted in final form 3 February 2014
fatty acid; glycerol; GC-MS; gene expression; lipogenesis; stable
isotopes
TRIGLYCERIDES (TG) make up 98% of the lipid content and, in
humans, contributes 40 –50% of the total energy content (28).
Total milk lipid content ranges, depending on the species, from
0 to 50% of the total milk volume (20). The fatty acid (FA)
composition of the TG is dependents on the dietary FA composition, the dietary carbohydrate/lipid ratio and the species.
Milk TG contains FA that are derived from three sources: de
novo mammary gland (MG) synthesis, dietary lipids, and
endogenous fat stores (adipose or hepatic lipids) (7). Data from
a study comparing the milk fats from seven different species
(4) revealed that the milk fats of humans, dogs, and guinea pigs
are largely (92, 93, and 97%, respectively) made up of longchain FA (LCFA). The milk fats of horses contain large
amounts (⬃33%) of medium-chain FA (MCFA); however, the
milk fats of cows, sheep, and goats are enriched (13–15%) in
short-chain FA (SCFA) (4). The MG is one of three primary
lipid-synthesizing organs in the body; the other two are liver
and adipose tissue (48). In fact, many of the initial pathways of
Address for reprint requests and other correspondence: M. W. Haymond,
Children’s Nutrition Research Center, 1100 Bates St., Houston, TX 77030
(e-mail: [email protected]).
E838
FA biosynthesis were defined using mammary tissue from
lactating ruminants and rodents (1). The unique feature of FA
synthesis in the MG is that saturated FA with 6 –14 carbons
constitutes the major product of de novo FA synthesis in these
animals (10, 38, 39). This is because the mammary alveolar
cells contain thioesterase II (OLAH), which terminates FA
synthesis after the addition of 8 –16 carbons (21). Thus, the
vast majority of longer-chain FA in milk are derived from the
diet or mobilization of endogenous TG (37). Generally, the rate
of TG synthesis in the lactating MG depends on the stage of
mammary development and is decreased by fasting and starvation in ruminants and rodents but not in species that fast
during lactation, such as seals and hibernating bears (14, 34). A
number of factors may play regulatory roles in the de novo FA
synthetic processes such as insulin, prolactin, and nonesterified
FA(32).
The FAs in milk TG are metabolized differently based on
their carbon chain length. SCFA and MCFA differ from LCFA
in that they are absorbed directly and rapidly oxidized (2),
induce satiety (40), and form precursors of important biological molecules (18, 33). MCFA in premature infants fed formulae containing medium chain triglycerides (MCT) have been
reported to increase absorption of calcium and magnesium (44)
and improve fat and nitrogen absorption (45). Therefore, the
possibility to alter favorably the composition of milk lipid
through dietary manipulation of maternal diet is appealing.
Hachey et al. (9) used 2H2O to measure the endogenous FA
synthesis of C10:0 to C18:0 saturated FA in both milk and
plasma TG during consumption of high-fat (H-FAT) and highcarbohydrate (H-CHO) diets in humans. Perhaps, due to the
relatively insensitive technique, they did not provide enrichments for either FA ⱕC10 in milk or FA ⱕC14 in plasma (9).
Additionally, enrichments of C16 and C18 were similar or
even higher in plasma compared with milk. As a result, they
were unable to determine whether these FA in milk were
synthesized within or were external to the MG or somewhere
else (liver, adipose tissue) and then transported to the MG to be
incorporated into milk (9).
In the present study, we used the incorporation of 13C from
[U-13C]glucose to measure de novo FA synthesis into specific
milk and plasma FA. In addition, we measured the incorporation of 13C into glycerol, the carbon backbone of TG, in both
plasma and milk. We applied this approach to study milk FA
composition and de novo TG synthesis from [U-13C]glucose in
blood and milk compartments following an overnight fast and
during continuous feeding of either a H-FAT or a H-CHO diet.
We hypothesized that de novo lipogenesis as reflected by the
incorporation of 13C carbons from [U-13C]glucose into FA and
glycerol in TG would be greater 1) in milk than in plasma TG,
2) during a H-CHO diet than a H-FAT diet under isocaloric and
isonitrogenous conditions, and 3) during feeding than during
fasting.
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Mohammad MA, Sunehag AL, Haymond MW. De novo synthesis of milk triglycerides in humans. Am J Physiol Endocrinol
Metab 306: E838 –E847, 2014. First published February 11, 2014;
doi:10.1152/ajpendo.00605.2013.—Mammary gland (MG) de novo
lipogenesis contributes significantly to milk fat in animals but little is
known in humans. Objective: To test the hypothesis that the incorporation of 13C carbons from [U-13C]glucose into fatty acids (FA) and
glycerol in triglycerides (TG) will be greater: 1) in milk than plasma
TG, 2) during a high-carbohydrate (H-CHO) diet than high-fat (HFAT) diet, and 3) during feeding than fasting. Seven healthy, lactating
women were studied on two isocaloric, isonitrogenous diets. On one
occasion, subjects received diets containing H-FAT or H-CHO diet
for 1 wk. Incorporation of 13C from infused [U-13C]glucose into FA
and glycerol was measured using GC-MS and gene expression in
RNA isolated from milk fat globule using microarrays. Incorporation
of 13C2 into milk FA increased with increased FA chain length from
C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was
not detected in FA⬎C16. During feeding, regardless of diets, enrichment of 13C2 in milk FA and 13C3 in milk glycerol were ⬃3- and
⬃7-fold higher compared with plasma FA and glycerol, respectively.
Following an overnight fast during H-CHO and H-FAT diets, 25 and
6%, respectively, of medium-chain FA (MCFA, C6 –C12) in milk
were derived from glucose but increased to 75 and 25% with feeding.
Expression of genes involved in FA or glycerol synthesis was unchanged regardless of diet or fast/fed conditions. The human MG is
capable of de novo lipogenesis of primarily MCFA and glycerol,
which is influenced by the macronutrient composition of the maternal
diet.
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
E839
MATERIALS AND METHODS
Materials
Procedure
Acetone, chloroform, ethanol hexane methanol, and all solvents
were HPLC grade. [U-13C]glucose (99 atom %); [13C3]- and
[2H5]glycerol (99 atom %); [13C3]lactate (98 atom %) and [1,213
C2]octanoic, myristic, and palmitic acids (98 atom %) were purchased from Cambridge Isotope Laboratory (Andover, MA).
O-(2,3,4,5,6-pentafluorbenyl) bromide (PFBBr) and tetrabutylammonium hydrogen sulfate (TBA) were obtained from Aldrich Chemicals
(Milwaukee, WI). FA standards (C2-C22) were obtained from SigmaAldrich (St. Louis, MO). The uniformly deuterium-labeled (99 atom
%), even-chain, saturated FA (C2:0, C4:0, C6:0, C8:0, C10:0, C12:0,
C14:0, C16:0, and C18:0) were obtained from Cambridge Isotope
Laboratory (Andover, MA) as internal standards. Details of FA
standards and their deuterated labeled internal standards has been
described previously (27).
Detailed information regarding subjects and procedures has already
been published (28). Briefly, following approval by the IRB at Baylor
College of Medicine and the General Clinical Research Center
(GCRC) Advisory Committee, written consent was obtained from
each of seven healthy, lean exclusively breast-feeding women, who
were 28.5 ⫾ 1.2 yr of age (mean ⫾ SE), weighed 60.0 ⫾ 1.0 kg, and
had a BMI of 22.2 ⫾ 0.7 kg/m2. At the time of these studies, their
babies were 10.0 ⫾ 2.0 wk of age and weighed 5.4 ⫾ 0.5 kg.
Protocol
Gene Expression Analysis
In milk samples from 9 AM and 4 PM of day 4 during each study
occasion, milk fat globule was collected and processed for gene
expression according to our previous reports (24, 26).
Analytical Methods
Substrate concentration. Glucose, lactose, and lactate in milk and
plasma were determined as previously reported (28). Free glycerol
concentration in milk and plasma was measured using reverse
isotope dilution methodology employing [2H5]glycerol as internal
standard.
Isotopic enrichments. GLUCOSE AND GLYCEROL. Enrichments of
13
C in the glucose isotopomers in plasma and milk were performed
using the acetic anhydride derivative (43). Similarly, the triacetate
derivative of glycerol was prepared using acetic anhydride. The
isotopic enrichments of glycerol isotopomers (M⫹1 to M⫹3) and the
internal standard [2H5]glycerol were measured by GC-MS (HP 5890/
HP5970; Hewlett-Packard, Palo Alto, CA) and an HP-1701 column
(30 m ⫻ 0.25 mm ⫻ 0.25 ␮m; Agilent Technologies, Wilmington,
DE). Positive chemical ionization mode using methane was used and
selected ion monitoring of m/z 159 –164 applied.
FA IN MILK AND PLASMA. The 13C enrichment and quantification of
FA in milk and plasma were performed on samples obtained during
the near steady state of tracer infusion (8 –9 AM and 3– 4 PM,
representing the fasting and fed states, respectively) using the PFBBr
derivatization and GC-MS negative chemical ionization (NCI) as
previously reported (27) with slight modification. In this study, we
utilized an Rtx-225 GC column (30 m ⫻ 0.25 mm ⫻ 0.25 ␮m; Restek,
Bellefonte, PA), allowing better peak shape and separation. The
conditions for the GC were as follows: injector: 250°C (splitless
injection of samples); oven: 60°C for 1.0 min; ramp, 15°C/min to
240°C; hold at 240°C for 10 min. In addition to better retention on the
GC column and higher resolutions and sensitivity, the PFBBr derivatives do not disrupt the natural isotopomer distribution in NCI,
because only the FA moiety is measured in NCI (47). The numerous
advantages of PFPBr derivative under NCI conditions, including
sensitivity and fragmentation pattern, enabled us to trace the isotopic
incorporation of 13C carbons from the [U-13C]glucose and to quantify
a wide spectrum of FA ranging from C2 to C22 in both plasma and
milk. Mass-to-charge ratios (m/z) of 89, 90, 91, and 92 were used to
monitor M⫹0, M⫹1, M⫹2, and M⫹3 for lactate, respectively.
Acetate enrichments and concentrations were performed similarly
using PFBBr derivative as previously described (27), with the exception that the samples were not subjected to the saponification process.
The same column and GC-MS condition modifications mentioned
above were also applied. The fragment masses of 59, 60, and 62 were
used to monitor M⫹0, M⫹1, M⫹2, and M⫹3. M⫹3 in this case
represents the internal standard d4-acetate, in which the deuterium on
the carboxyl group is being lost during derivatization. All measurements were made in the Stable Isotope Core Laboratory of the
Children’s Nutrition Research Center.
RNA isolation, cRNA amplification and expression microarray.
Total RNA was isolated from TRIzol-treated milk fat as previously
described (26). The methods utilized for cRNA amplification and
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Each woman was studied on two occasions for 8 days each
separated by 1–2 wk. On each study occasion, the subjects were
admitted to the GCRC for 4 days and 3 nights. The subjects were
randomly assigned to receive either a low-carbohydrate/high-fat diet
(H-FAT: 30% carbohydrate, 55% fat, and 15% protein) on one
occasion or a high-carbohydrate/low-fat diet (H-CHO: 60% carbohydrate, 25% fat, and 15% protein) on the other occasion. Total energy
intake for each woman was set as a multiple of 1.3 times basal
metabolic rate (BMR). For 4 days before admission, 3 meals and 3
snacks/day were weighed, prepacked, and sent to the subject’s home
by our research kitchen [an example of a 1-day menu of the diets has
been presented previously as supplemental material (28)]. Following
the 4-day diet period at home, the subjects were admitted at ⬃4 PM
to the GCRC for 4 days and 3 nights. During the first 3 days, the
subjects received the same intake and diet composition as that consumed at home. During the stay in the GCRC, women breastfed their
babies every 3 h and expressed 2.5 ml of milk from each breast at the
beginning, middle, and after the feeding (a total of 15 ml each
feeding). Milk samples for each collection time were pooled for milk
analyses. The babies were weighed before and after each breastfeeding to estimate the milk volume consumed (16). After breastfeeding,
the mothers again were asked to empty their breasts completely using
a breast pump. This extra milk was weighed. The milk volume
produced during each feeding was calculated from the sum of the
delta in the baby’s weight over each feeding plus the pumped milk.
On the evening of day 3, the subjects were provided dinner at 6 PM
and a snack at 8 PM. Thereafter, except for water, they were fasted
until 9 AM the following morning (day 4). In the evening of study day
3, two intravenous catheters were placed under ELAMAX cream
analgesia (Astra Pharmaceuticals, Wayne, PA). One was placed in an
antecubital vein for isotope infusion and a second in a vein in the
contralateral arm for blood sampling. At 4 AM on study day 3, a
primed constant infusion of [U-13C]glucose (61 ⫾ 0.7 ␮mol/kg and
1.02 ⫾ 0.01 ␮mol·kg⫺1·min⫺1) was initiated to measure glucose rate
of appearance and glucose production from gluconeogenesis (28).
From 9 AM until 4 PM, the women consumed small, frequent meals
(q15 min). The food had the same composition and provided the same
calories as that consumed on a daily basis over the previous week. At
the initiation of the feedings, a second priming dose of [U-13C]glucose
(161 ⫾ 4 ␮mol/kg) was given, and the infusion rate was increased to
2.7 ⫾ 0.1 ␮mol·kg⫺1·min⫺1 to maintain sufficient [U-13C]glucose
enrichment to measure accurately the rate of gluconeogenesis (28). At
3 AM, a baseline blood sample (10 ml) was obtained. Subsequently,
5 ml of blood was drawn every 15 min from 8 AM to 9 AM and from
3 PM to 4 PM. Milk samples were obtained at 3 AM (baseline), 9 AM,
12 PM, and 4 PM.
E840
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
microarray expression analyses were identical to that previously
validated using RT-PCR and published (24, 25), using human Ref-8
BeadChips and the BeadStation system from Illumina (San Diego,
CA). After scanning of the probe array, the resulting image was
analyzed using the GenomeStudio software (Illumina). Details about
raw intensity data analysis and normalization were discussed in our
previous publication (24). These data have been deposited in NCBI’s
Gene Expression Omnibus (6) and are accessible through GEO Series
accession number GSE51874 (http://www.ncbi.nlm.nih.gov/geo/
query/acc.cgi?acc⫽GSE51874).
Calculations
The precursor product relationship has been utilized to calculate de
novo synthesis of both FA and glycerol.
de novo glycerol synthesis (%)
⫽
M⫹3 enrichment of glycerol ⫻ 100
M⫹6 enrichment of glucose
Total amount of labeled FA was calculated using the following
formula:
⫽
16
兺2 关共M ⫹ 2)i ⫻ Ci兲兴
兺
16
where
2 is the sum of total labeled FA (mg/l), i represents an
even-chain FA of 2–16 carbons, M⫹2 is the enrichment of FAi (%),
and C is the concentration of the FAi (mg/l). Total amount of labeled
free glycerol was calculated by multiplying [M⫹3]glycerol enrichment with the free glycerol concentration.
Statistical Analysis
Data were averaged for each subject during each study occasion
and the mean ⫾ SE was calculated for each diet occasion. A paired
Student’s t-test was used to compare the effect of the two diets on
the average of variables measured on day 4 during the fasting (8 –9
AM) and feeding (3– 4 PM) states. The Benjamini-Hochberg false
discovery rate (B-H FDR) correction for multiple analyses following paired Student’s t-test was used to compare the gene expression
between diets and fasting/feeding conditions. Significance was set
as P ⬍ 0.05.
RESULTS
Data related to dietary intakes, plasma substrate concentrations, milk volume and composition, infant intakes, glucose
kinetics, energy expenditure, and substrate oxidations are presented in our previous publication (28).
FA Concentrations
Following overnight fasting, the concentration of C10:0 FA
in plasma was lower (P ⬍ 0.01) in the H-CHO than in the
H-FAT diet (Table 1). During feeding, plasma C2:0, C16:0,
C18:0, C18:1, C18:2, and C20:4 and sum of total FA were
higher (P ⬍ 0.05) during the H-FAT diet than in the H-CHO
diet. The concentration of individual and sum of C4 –C14 were
all higher (P ⬍ 0.01) in milk in the H-CHO compared with the
H-FAT diet during both feeding and fasting (Table 1). However, the concentrations of C18:1, C20:2, C20:3, C20:4, C20:2,
and C22:6 were all higher during the H-FAT diet following the
overnight fasting. Similarly, C16:0, C18:0, C18:1, C18:2, and
Isotopomer Enrichments
Following overnight fasting. M⫹6 enrichment of glucose
was slightly higher (P ⬍ 0.05) in both plasma and milk during
the H-FAT diet compared with H-CHO diet and lower (P ⬍
0.01) in milk compared with plasma during both diets (Fig. 1).
M⫹3 enrichment of lactate was not different between the two
diets in either plasma or milk but was higher (P ⬍ 0.01) in
plasma compared with milk regardless of the diet (Fig. 1). The
primary isotopomer detected in all FA (C2–C16) was M⫹2
(Fig. 1), with little contribution in M⫹1 or M⫹3 in FA ⱖ4C
(data not shown). Enrichment of M⫹2 acetate was not different between the diets or between milk and plasma (Fig. 1).
M⫹2 enrichments of C4 –C10 FA in both plasma and milk
were higher (P ⬍ 0.05) during the H-CHO diet than in the
H-FAT diet. However, the M⫹2 enrichments of C4 –C10 FA
were lower (P ⬍ 0.05) in milk compared with plasma under
both dietary conditions (Fig. 1). M⫹2 enrichment of C12 FA
in plasma and milk was higher in the H-CHO than in the
H-FAT diet and higher (P ⬍ 0.05) in milk compared with
plasma during both diets (Fig. 1). M⫹2 enrichment of C14 FA
in milk was higher in the H-CHO than in the H-FAT diet and
higher (P ⬍ 0.05) in milk compared with plasma during both
diets (Fig. 1). Little M⫹2 enrichment was observed in palmitate but was similar between the diets and between milk and
plasma (Fig. 1).
During feeding. M⫹6 enrichment of glucose was higher
(P ⬍ 0.01) during the H-FAT diet in both plasma and milk but
lower (P ⬍ 0.01) in milk compared with plasma during both
diets (Fig. 1). M⫹3 enrichment of lactate was higher in plasma
during the H-FAT diet compared with H-CHO diet and was
higher (P ⬍ 0.01) in plasma compared with milk during both
diets. M⫹2 acetate was not different between the two diets and
between milk and plasma. M⫹2 enrichments of the FA C4 –
C14 in plasma were similar between the two diets (P ⬎ 0.05),
but all were higher in milk (P ⬍ 0.05) during the H-CHO diet
compared with H-FAT diet and were higher (P ⬍ 0.05) in milk
compared with plasma regardless of the diet. M⫹2 enrichment
of C12 FA in plasma and milk was higher during the H-CHO
compared with the H-FAT diet and higher (P ⬍ 0.05) in milk
compared with plasma during both diets. M⫹2 enrichment of
C14 FA in milk was higher during the H-CHO compared with
the H-FAT diet and higher (P ⬍ 0.05) in milk compared with
plasma during both diets. M⫹2 enrichment of palmitate was
higher (P ⬍ 0.05) in milk during the H-CHO compared with
the H-FAT diet during feeding and was higher (P ⬍ 0.05) in
milk compared with plasma during the H-CHO diet only.
FA De Novo Synthesis from Glucose
Following overnight fasting. De novo synthesis of C4 –C10
in plasma was higher (P ⬍ 0.01) during the H-CHO (10 – 45%)
compared with H-FAT diet (7–15%; Fig. 2). In milk, %de novo
synthesis of C4 –C14 were higher (P ⬍ 0.01) during the
H-CHO (20 –35%) compared with the H-FAT (7–15%) diet
(Fig. 2). During the H-CHO diet, %de novo synthesis of C6
and C10 was higher, but those of C12 and C14 were lower (P ⬍
0.01) in plasma compared with milk (Fig. 2).
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de novo FA synthesis (%)
M⫹2 enrichment of any given fatty acid ⫻ 100
⫽
M⫹2 enrichment of acetate ⫻ number of C2 units in the FA
C18:3 were higher during feeding in milk, reflecting the FA
composition of the diet (Table 1).
E841
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
Table 1. Total and individual plasma (Pl) and milk (Mk) fatty acid composition during fasting and feeding of high
carbohydrate (HC) and high fat (HF) diets in lactating women
Overnight Fasting
Pl-HC
Pl-HF
Mk-HC
Feeding
Mk-HF
Pl-HC
Pl-HF
Mk-HC
Mk-HF
2.919
0.008
0.003
0.003
0.004
0.008
0.016
0.055
0.097
0.411
0.232
0.740
0.094
0.556
0.046
0.696
0.792
0.069
0.028
0.128
0.322
0.008
0.043
0.092
1.483
3.411*
0.010*
0.002
0.002
0.004
0.010
0.015
0.053
0.097
0.483**
0.285**
0.865*
0.082
0.625*
0.050
0.757
1.060**
0.064
0.025
0.097
0.378*
0.009
0.051
0.105
1.788**
42.239
0.008
0.148
0.333
0.530
1.920
3.569
3.915
10.423
4.846
2.944
18.212
2.143
10.110
1.118
13.371
6.317
1.268
0.493
0.657
1.020
0.083
0.234
0.582
10.655
46.893*
0.009
0.087*
0.183*
0.381**
1.547*
3.078*
3.427**
8.712**
5.620*
4.034**
18.366
1.760
13.230**
1.018
16.008**
7.595**
1.836**
0.532
0.563
1.173
0.056
0.229
0.535
12.519*
24.699
43.139
31.753
25.108
18.613**
39.142*
34.289*
26.569
g/l
2.828
0.010
0.001
0.001
0.002
0.002
0.006
0.046
0.069
0.417
0.230
0.716
0.100
0.558
0.048
0.706
0.746
0.067
0.027
0.110
0.318
0.008
0.038
0.091
1.406
2.815
0.011
0.003
0.003
0.004
0.005*
0.009
0.044
0.078
0.409
0.235
0.722
0.081
0.555
0.051
0.687
0.798
0.056
0.020
0.081
0.320
0.008
0.038
0.085
1.407
41.908
0.007
0.102
0.202
0.357
1.313
2.618
2.632
7.230
5.326
3.157
15.713
2.040
11.408
1.303
14.751
6.678
1.403
0.598
0.616
1.120
0.106
0.261
0.664
11.445
40.833
0.008
0.071*
0.131**
0.246**
1.112*
2.383
2.320**
6.269**
5.456
3.308
15.033
2.125
12.748
0.909
15.783
6.471
1.335
0.439*
0.372*
0.820*
0.024*
0.148
0.408*
10.017
%Total
冱C2:C14
冱SAT
冱MUFA
冱PUFA
2.421
25.317
24.959
49.724
2.744
25.656
24.515
49.829
17.096
37.198
35.459
27.342
15.054*
36.230
38.962*
24.809
3.280
25.343
23.841
50.817
2.822
25.415
22.187*
52.398
Values are means from 7 subjects. SAT, saturated FA, including C2:C14; MUFA, monounsaturated FA and PUFA polyunsaturated FA. Significantly different
from H-CHO (paired t-test): *P ⬍ 0.05, **P ⬍ 0.01. Obviously in milk, except for C2, concentration of individual and sum of FA groups are severalfold higher
(P ⬍ 0.01) than the respective values in plasma.
During feeding. De novo synthesis of C4 –C16 FA was not
different between diets in plasma but in milk was ⬃2.5 times
higher (P ⬍ 0.05) in all FA (C4 –C12) during the H-CHO
(⬃75%) compared with the H-FAT diet (⬃30%). Similarly, de
novo FA synthesis was higher (P ⬍ 0.01) in C14:0 FA (50 vs.
20%) and C16:0 FA (10 vs. 5%) in milk during the H-CHO
diet compared with the H-FAT diet. In plasma, total amount of
labeled FA was ⬍1.5 mg/l, with no difference between diets
regardless of whether fed or fasting (Fig. 4). The total labeled
FAs in milk were higher in the H-CHO compared with the
H-FAT diet during both fasting (317 ⫾ 70 vs. 89 ⫾ 26 mg/l,
P ⬍ 0.01) and feeding (1,186 ⫾ 240 vs. 326 ⫾ 90 mg/l, P ⬍
0.01) compared with the H-FAT diet.
Glycerol Concentration, Enrichment, and De Novo
Synthesis
Generally, free glycerol concentrations were 5- to 10-fold
higher (P ⬍ 0.01) in milk compared with plasma during
either feeding or fasting (Fig. 3). Following an overnight
fast, the concentration of free glycerol was not different
between diets in either plasma or milk (Fig. 3). During
feeding, glycerol concentrations were lower (P ⬍ 0.05)
during the H-CHO diet in plasma but higher (P ⬍ 0.01) in
milk compared with the H-FAT diet (Fig. 3). Plasma en-
richment of M⫹3 glycerol in milk was higher (P ⬍ 0.01)
than that of plasma during both the fast (5-fold higher) and
fed (10-fold) conditions regardless of diet. Plasma enrichment (%) of M⫹3 glycerol was not different between
H-CHO and H-FAT diets during either fasting or feeding
(Fig. 1). However, milk M⫹3 glycerol was higher (P ⬍
0.01) in the H-CHO compared with the H-FAT diet during
both fasting and feeding. De novo synthesis of glycerol in
plasma was higher (P ⬍ 0.05) in the H-CHO diet compared
with the H-FAT diet during fed condition only (3.24 ⫾ 0.59
vs. 2.05 ⫾ 0.29, P ⬍ 0.05; Fig. 3). However, during the
H-CHO diet compared with the H-FAT diet, de novo synthesis of glycerol in milk (%) was higher during both fasting
(15.20 ⫾ 1.24 vs. 9.36 ⫾ 0.91, P ⬍ 0.01) and feeding (35.55 ⫾
1.36 vs. 17.66 ⫾ 1.76, P ⬍ 0.01) (Fig. 3).
Total amount of labeled free glycerol was ⬍2.5 mg/l in
plasma, but higher (P ⬍ 0.01) in the H-FAT compared with the
H-CHO diet during feeding only (Fig. 4). Total labeled free
glycerol in milk was not different between diets during fasting
(23 ⫾ 5 vs. 19 ⫾ 6 mg/l, P ⬎ 0.05). However, during feeding,
total labeled free glycerol was ⬃4-fold higher in the H-CHO
(317 ⫾ 70 vs. 89 ⫾ 26 mg/l, P ⬍ 0.01) compared with the
H-FAT diet. Obviously, feeding increased (P ⬍ 0.01) the
amount of labeled glycerol compared with fasting in both
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Total
C2:0
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
冱C2:C14
C16:0
C18:0
冱SAT
C16:1
C18:1
C20:1
冱MUFA
C18:2
C18:3
C20:2
C20:3
C20:4
C20:5
C22:5
C22:6
冱PUFA
E842
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
Enrichment
Plasma
15.0
Fasting
Percent
12.5
H-CHO
H-FAT
10.0
*
7.5
5.0
2.5
** ** **
0.0
15.0
**
Feeding
*
H-CHO
H-FAT
12.5
Percent
Fasting
H-CHO
HH-FAT
** ** ** ** **
**
Feeding
**
H-CHO
H-FAT
10.0
**
7.5
**
**
5.0
**
2.5
**
** **
**
*
plasma and milk, but the magnitude of increase was greater in
milk (Fig. 4).
:0
:0
C
16
:0
14
12
C
C
0
:0
10
C
0
C
8:
0
6:
4:
C
C
G
lu
ce
r
La
ct
C
2:
0
ly
G
G
ly
G
lu
ce
r
La
ct
C
2:
0
C
4:
0
C
6:
0
C
8:
0
C
10
:0
C
12
:0
C
14
:0
C
16
:0
0.0
member 2 (THEM2) were not different between diets during
either feeding or fasting or when compared between fed and
fasted states within each diet (Fig. 5). Expression of the genes
for sterol regulatory element-binding transcription factor 1
(SREBF1), estrogen receptor 1 (ESR1), thyroid hormoneresponsive protein (THRSP), insulin-induced 2 (INSIG2), and
peroxisome proliferator-activated receptor-␥ (PPARG) were
not different regardless of diet or absorptive state (data not
presented). Similarly, expressions of genes for glycolysis or
Gene Expression Results
The mRNA expression of genes known to be involved in the
de novo FA synthesis including ATP citrate lyase (ACLY),
citrate synthase (CS), and transporter (SLC25A1), acetyl-CoA
carboxylase-␣ (ACACA) and -␤ (ACACB), FA synthase
(FASN), thioesterase 2 (OLAH), and thioesterase superfamily
FA de novo synthesis
100
Milk
Plasma
Fasting
Percent
H-CHO
H-FAT
50
**
25
*
**
**
**
**
0
100
**
*
*
**
Feeding
Feeding
H-CHO
H-FAT
H-CHO
H-FAT
75
Percent
Fig. 2. De novo synthesis of FA (%) in
plasma (left) and milk (right) pools during
infusion of [U-13C]glucose (and based on
acetate M⫹2 enrichments as a surrogate for
cytosolic acetyl-CoA enrichment) following
overnight fast (top) and during feeding (bottom) of H-CHO and H-FAT diets. Values
are means ⫾ SE; n ⫽ 7. Significantly different from H-CHO (paired t-test): *P ⬍ 0.05,
**P ⬍ 0.01.
Fasting
HH-CHO
H-FAT
H-
75
50
**
**
**
**
**
**
25
*
0
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C16:0
C4:0
C6:0
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C8:0
C10:0
C12:0
C14:0
C16:0
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Fig. 1. Enrichment of Glu, [M⫹6]glucose;
Glycer, [M⫹3]glycerol; Lact, [M⫹3]lactate,
and [M⫹2] in different FA in plasma (left)
and milk (right) during infusion of [U-13C]glucose following overnight fast (top) and
during feeding (bottom) of high-carbohydrate/low-fat (H-CHO) and low-carbohydrate/
high-fat (H-FAT) diets. Values are means ⫾
SE; n ⫽ 7. Significantly different from HCHO (paired t-test): *P ⬍ 0.05, **P ⬍ 0.01.
Milk
E843
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
Plasma
Glycerol concentration
1000
Milk
H-CHO
H-FAT
H-CHO
HH-FAT
µM/L
750
**
500
250
*
0
Fasting
Feeding
H-CHO
H-FAT
H-CHO
H-FAT
**
20
**
10
*
0
Fasting
Feeding
Fasting
glycerol kinases associated with glycerol synthesis and its
incorporation into the TG backbone were unchanged (data not
presented).
DISCUSSION
We have previously reported (28) that the total milk fat
concentration was ⬃13% higher (4.8 ⫾ 0.3 vs. 4.3 ⫾ 0.3 g/dl,
P ⬍ 0.05) and daily milk fat output was ⬃15% higher (39 ⫾
2 vs. 34 ⫾ 2 g/day, P ⬍ 0.05) during the H-FAT diet compared
with the H-CHO diet. However, the present study showed that
maternal dietary macronutrient composition, under isocaloric
isonitrogenous conditions, influences milk lipid composition
and that the human MG responds to dietary macronutrient
manipulation by altering the de novo synthesis of MCFA and
glycerol in milk. Whether expressed as concentration (g/dl) or
percentage of total, the individual and the sum of FA (C6 –
C14) were higher during feeding of the H-CHO diet compared
with the H-FAT diet. These findings are consistent with early
reports in four ethnic groups in East Africa and the Middle
East, in which the authors attributed the high concentrations of
lauric (C12:0) and myristic (C14:0) acids in milk (30% of total)
to their high-carbohydrate diet (35). Similar findings were also
reported in humans by other investigators (8, 9, 13, 15). In
other animal species, the MG is known to decrease FA synthesis in response to a high-fat diet (23, 32, 36).
The detection of 13C incorporation (as M⫹2) as the main
isotopomer in both plasma and milk FA confirms the known
role for acetyl-CoA as a precursor for FA synthesis. The
detection of M⫹2 and its enrichment pattern in both plasma
and milk indicate that human tissues are capable of significant
de novo synthesis of SCFA and MCFA (C4 –C14) but insignificant enrichment of FA ⱖC16. Detection of M⫹2 enrich-
Feeding
ment in FA with 16 or fewer carbons in the plasma compartment may indicate that tissues other than the MG (most likely
liver) contribute to the de novo FA synthesis process, assuming
little or no FA escape the MEC and enter the circulation. The
higher the M⫹2 enrichments of FA with 10 or fewer carbons
in plasma compared with milk following an overnight fast may
indicate a greater contribution from these tissues in this de
novo synthetic process. Whether these FA are utilized by
tissues as fuel and/or are specifically transported to the MG
remains to be determined. Yet the concentrations of these FA
in plasma are very low (⬍1/40) compared with those of milk,
and consequently their higher enrichment may be due to the
smaller pool size in plasma vs. milk. The higher M⫹2 enrichments of C12:0 and C14:0 in milk compared with plasma
(regardless of diet or fasting/feeding conditions) and their
relatively higher abundance in milk support the contention that
the MG may be the primary site for synthesis of these two FAs.
Generally, the higher M⫹2 enrichment in milk FA compared
with plasma reflects the increase in the de novo synthesis
during feeding condition. However, the higher M⫹2 enrichments and concentrations of these FA in both plasma and milk
during the H-CHO diet indicate increased de novo synthesis
induced by the carbohydrate diets. The inability to detect
significant enrichments in FA longer than 16 carbons in either
milk or plasma indicates little or no de novo synthesis of these
FAs by the human tissues and/or tremendous dilution by the
dietary and mobilized FAs under our study conditions.
The pattern of 13C labeling from our study is in general
agreement with the findings from MG of lactating goats infused with substrates labeled with 14C. These studies demonstrated extensive 14C labeling of milk FA with chain length up
to C(14) and to a smaller extent for the synthesis of palmitate
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Percent
Fasting
Glycerol de novo synthesis
40
30
Feeding
Fig. 3. Concentration(␮M; top) and de novo
synthesis of free glycerol (%; bottom) in
plasma and milk pools following overnight
fast and during feeding of H-CHO and HFAT diets and infusion of [U-13C] glucose.
Values are means ⫾ SE; n ⫽ 7. Significantly
different from H-CHO (paired t-test): *P ⬍
0.05, **P ⬍ 0.01.
E844
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
Plasma
1.75
1.50
1750
1500
HH-CHO
H-FAT
1250
1.00
1000
0.75
750
0.50
500
0.25
250
0.00
Fasting
Feeding
H-CHO
H-FAT
**
**
0
Fasting
Feeding
Absolute labeled free glycerol
4.00
400
H-CHO
HH-FAT
mg/L
3.00
**
300
2.00
200
1.00
100
0.00
Fasting
(22). Similar findings have been reported in rabbits (5) in
which the specific radioactivity of the individual FA (C6:0 to
C14:0) and the proportions of these FAs synthesized were
similar in mammary tissue and milk. Hexanoic acid (C6:0) had
the highest specific radioactivity, and the C8:0 to C14:0 FAs
had similar specific radioactivities, which were about five
times those of C16 and C18 acids. No radioactivity was
detected in FA of chain length ⬍C14 in the liver, blood, or
adipose tissue, and the specific radioactivities of FA of chain
length ⬎C14 in these tissues were similar to those of the LCFA
in the milk and MG. The results demonstrated that the C4:0 to
C14:0 FA are synthesized within the MG rather than the result
of FA uptake from circulating blood or by partial or complete
oxidation of LCFA within the MG. However, mammary slices
from lactating guinea pigs synthesize LCFA (C16 –C18, which
are the predominant acids of guinea pig milk) saturated and
unsaturated FA from acetate in the presence or absence of
glucose (41). Cells from bovine lactating MG synthesized
mostly SCFA and MCFA (C4 –C12), whereas the rat mammary cells synthesized FA ranging from C6 to C18 (17).
Measurements of de novo lipogenesis are of great interest
with the epidemic of obesity and its related comorbidities.
Tracer techniques are required to distinguish between dietary
and newly synthesized FA. To measure the fractional de novo
synthesis of FA, the enrichment of the true precursor of FA
synthesis (cytosolic acetyl-CoA) is required, and several methods have been attempted to do so, including sampling of the
lipogenic hepatic acetyl-CoA pool in vivo in rats by using a
xenobiotic probe (12) and prediction from isotopomeric distribution in circulating lipids and measurement of lipogenesis and
acetyl-CoA dilution (11). In our study, we attempted to utilize
the enrichment of acetate as a surrogate for intracellular acetylCoA. Although acetate (in milk or plasma) may not reflect the
true cytosolic acetyl-CoA pool, we believe it gives an estimate
Feeding
H-CHO
H-FAT
0
**
Fasting
Feeding
of the intracellular acetate enrichment for the fractional de
novo synthesis calculations to compare change in FA synthesis
under our fasting and fed conditions using a paired study
design. Nevertheless, our calculations showed that the de novo
synthesis reached as high as 75% of FA with C4 –C12 during
the consumption of the H-CHO diet, which is almost 2.5 times
greater than those obtained during the H-FAT diet (⬃30%). In
milk, the consistent values for de novo synthesis among these
FAs (C4 –C12) indicated that these FAs originated from a
common precursor pool. However, in plasma, inconsistencies
were observed for the values of de novo synthesis among
C4 –C12 FAs, indicating that they most likely had arisen from
different precursor pools. Another possible explanation, as
described above, is the trivial pool size of these circulating
FAs. This might be further supported by the calculated absolute labeled FA in plasma compared with milk (Fig. 4). The
decrease in the percent de novo synthesis in C14:0 in milk and
longer-chain FAs may be explained by the increased contribution from the dietary and endogenously mobilized FA. Our
data and those of Hachey et al. (9) are in good agreement with
the in vitro studies of human MECs isolated from breast milk,
indicating that the human MG has the ability to synthesize
C10:0, C12:0, and C14:0 FAs but has a limited capacity to
synthesize C16:0 and C18:0 (46).
During the secretory activation phase, changes in FA composition were mirrored in the expression of the genes for the
enzymes involved in de novo FA synthesis within the MG (27).
However, gene expression was unaltered by dietary macronutrient composition in the present study. This suggests that the
substrate availability and/or change in circulating hormones
(e.g., insulin) may be regulating de novo FA synthesis independently of the change in gene expression. In this regard, we
previously reported (28) that circulating insulin concentrations
were higher during the H-CHO diet compared with the H-FAT
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Fig. 4. Absolute amount of sum of labeled FA
(top; as calculated by multiplying the enrichment of M⫹2 FAs times their concentrations)
and free glycerol (bottom; as calculated by
multiplying the enrichment of M⫹3 glycerol
times its concentration) in plasma and milk
following overnight fast and during feeding of
H-CHO and H-FAT diets. Note the y-axis in
the right is 1,000 (top) and 100 (bottom) times
that in the left. Values are means ⫾ SE; n ⫽
7. Significantly different from H-CHO (paired
t-test): **P ⬍ 0.01.
mg/L
1.25
Milk
Absolute labeled FAs
E845
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
Fluorescence Intensity
CS
4000
SLC25A1
1250
H-CHO
H-Fat
ACACA
1500
10000
1000
5000
500
H-CHO
H-Fat
1000
3000
750
2000
500
1000
0
250
Fast
Fed
0
ACACB
10000
H-CHO
H-Fat
7500
Fast
Fed
0
Fast
FASN
0
Fed
OLAH
50000
25000
40000
20000
30000
15000
20000
10000
10000
5000
Fast
Fed
THEM2
2000
H-CHO
H-Fat
1500
5000
1000
2500
0
0
Fast
Fed
Fast
Fed
0
Fig. 5. Fluorescence intensity of genes involved in de novo FA synthesis in milk samples following overnight fast and during feeding of H-CHO and L-CHO diets. CS, citrate
synthase; SLC25A1, solute carrier family 25
[mitochondrial carrier; citrate transporter 1;
ACLY, ATP citrate lyase; ACACA, acetylCoA carboxylase-␣; ACACB, acetyl-CoA
carboxylase-␤; FASN, FA synthase; OLAH,
S-acyl FA synthase thioesterase, medium
chain (thioesterase II or oleoyl-acyl-carrier
protein hydrolase); THEM2, thioesterase superfamily member 2]. Values are means ⫾
SE; n ⫽ 7. None of the genes (P, paired t-test;
B-H FDR for multiple corrections, ⬎0.05)
was different between diets or fast/fed conditions.
500
0
Fast
Fed
diet during both fasting and feeding. However, our recent
studies elucidate that the major glucose transport systems in the
human MECs are insulin-independent transporters (26), excluding a role for insulin in facilitating glucose transport into
the MEC. Accordingly, insulin may be exerting an indirect
effect by increasing the activity of acetyl-CoA carboxylase
(ACC) by decreasing its phosphorylation (31), thus increasing
de novo FA synthesis. This mechanism is based on two
independent observations: 1) the ability of insulin to stimulate
MG FA synthesis as a consequence of 24 h of food deprivation
in rats (49), and 2) refeeding with and without inhibition of
insulin release using streptozotocin (30). In addition, insulin’s
inhibition of lipolysis in lactating women (29) may support the
notion that NEFA themselves regulate de novo MG FA synthesis by inhibition of the activity of ACC (32). Consistent
with this speculation is the observation in these women that
NEFA and ketones were higher during the H-FAT diet than
during the H-CHO diet (28).
Prolactin has long been considered a primary regulator of the
synthesis of milk components including lipids. However, we
did not find any difference in circulating prolactin concentrations between the two diets under either fasting (99 ⫾ 18 vs.
85 ⫾ 16 ␮U/ml, P ⫽ 0.40) or fed (45 ⫾ 15 vs. 62 ⫾ 20 ␮U/ml,
P ⫽ 0.11) conditions. Additionally, genes on the prolactin
receptor and its downstream pathway, including SREBF1 and
others related to de novo synthesis (26, 27), were not different
regardless of diet or fast/fed states. Based on our limited
sample size and microarray data, these observations exclude
the role of prolactin in this process and may imply that the
effects of this hormone may be related more to the establishment of lactogenesis and involution than to moment-to-moment regulation of the rate of synthesis of milk components
(26, 27, 32). However, these findings may require further
validation.
Data related to gene expression from our study are in
agreement with those obtained from the MG of mice fed diets
Fast
Fed
containing either 8 or 40% of their calories as lipid, substituting lipid for carbohydrate, from days L5 to L10, using Affymetrix gene chips (36). Those authors found that expression
of only 85 genes (excluding those involved in de novo synthesis) was significantly altered by the high-fat diet in the MG
compared with 760 in the liver (36). By comparison, the
lactating MG is devoted to one process, the synthesis and
secretion of milk (32). It worth noting that fat in rodent milk is
fivefold higher in concentration than human milk (20 vs. 4% of
total weight). However, our data are not consistent with those
from dairy cows, in which a total of 972 genes were differentially expressed in the MG tissue when the grazing dairy cow
diet was supplemented with unsaturated FA (UFA) compared
with cows fed a control diet. This suggests a large degree of
transcriptomic adaptation to the dietary UFAs (23) in the cow.
Our data show that the human MG can significantly synthesize glycerol from glucose (glyceroneogenesis), accounting for
⬃15 and 30% of milk glycerol during fasting and fed conditions, respectively. Indeed, this feature of the MG would meet
the higher demand of glycerol required for milk TG synthesis
(⬃30 mM). We speculate that the capability of MEC to
efficiently synthesize glycerol from glucose is the result of
expression of several genes in the glycolytic, pentose phosphate shunt, and glycerol activation pathways (26, 27), all of
which are upregulated during the secretory activation phase of
lactation. However, as observed with genes involved in FA
synthesis, in the present study expression of genes associated
with glycerol synthesis and its incorporation into the TG
backbone was unaltered by dietary macronutrient composition.
In contrast, non-MG tissues, compared with MG, appeared to
have limited capabilities to de novo synthesize of glycerol (Fig.
3). The effect of the diet was prominent, however, in that the
de novo synthesis of glycerol was high in milk during the
H-CHO diet compared with the H-FAT during both fed and
fasting conditions. Obviously, during the H-FAT diet lipolysis
of the dietary fat during meal absorption and mobilization of
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Fluorescence Intensity
ACLY
15000
E846
DE NOVO SYNTHESIS OF MILK LIPIDS IN HUMANS
ACKNOWLEDGMENTS
We thank volunteers whose participation made this study possible. We
gratefully acknowledge and thank the technicians in our laboratory (Dr. Susan
Sharma, Marcia Ekworomadu, Dr. Shaji Chacko, and Dan Donaldson), our
research coordinators (Amy Pontius, Cindy Bryant, and Linda Pleasant), and
the staff in the Metabolic Research Unit and kitchen and the General Clinical
Research Center who greatly facilitated the execution of these studies.
GRANTS
This project was supported by NIH Grants RO1 DK-55478, HD-37857,
MO1 RR-00188, USDA/ARS 6250-5100. This work is a publication of the
USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics,
Baylor College of Medicine (Houston, TX). The contents of this publication do
not necessarily reflect the views of policies of the US Dept. of Agriculture, nor
does mention of trade names, commercial products, or organizations imply
endorsement from the US Government.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.A.M., A.L.S., and M.W.H. conception and design
of research; M.A.M. performed experiments; M.A.M. analyzed data; M.A.M.,
A.L.S., and M.W.H. interpreted results of experiments; M.A.M. and M.W.H.
prepared figures; M.A.M. drafted manuscript; M.A.M., A.L.S., and M.W.H.
edited and revised manuscript; M.A.M., A.L.S., and M.W.H. approved final
version of manuscript.
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stored TG following an overnight fast (possibly due to lower
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The possibility of altering favorably the composition of milk
lipid through dietary manipulation of maternal diet is interesting. Glycerol is a very important nutrient in breast milk,
representing the second most abundant molecule (based on
molar distribution, ⬃40 mM) after lactose. In human newborns, ⬃75% of transported glycerol was converted to glucose
and represented 5.0% of hepatic glucose production (3); however, glycerol accounts for ⬃60% of the substrate for total
gluconeogenesis in very low birth weight infants receiving
TPN (42). The FA in milk TG are metabolized differently
based on their carbon chain length. MCFA differ from LCFA
in that they are absorbed directly into the portal circulation and
the vast majority are taken up by the liver on a first-pass basis
and are rapidly oxidized (2). MCFA have been reported to
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(18) and ghrelin (C12:0) (33). Finally, premature infants fed
MCT-containing formulae, when compared with controls, absorbed more calcium and magnesium (44) and improved fat
and nitrogen absorption (45).
In summary our data show that alterations in the macronutrient composition of the maternal diet affect the production of
selected FA and the lipid composition of the human milk and
illustrate the complex relationships among diet, energy metabolism, and milk composition. The impact of these changes in
milk lipid composition on infant nutrition and metabolism will
require further investigation.
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