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Department of Pediatrics, Children’s Nutrition Research Center, United States Department of Agriculture/Agricultural Research Service, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: M. W. Haymond, M.D., CNRC, 1100 Bates Street, Houston, Texas 77030-2600. E-mail: mhaymond{at}bcm.tmc.edu
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Abstract |
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Introduction |
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Little or no information is available concerning the metabolic adaptation of lactating mothers to either feeding or short-term fasting. In the fed circumstance, this could be met by 1) increasing the carbohydrate intake; 2) decreasing the normal suppression of endogenous glucose production; 3) reducing splanchnic extraction of dietary glucose; or 4) reducing glucose oxidation and/or storage. However, it is not known how a lactating mother would metabolically adapt were she unable to meet this increased nutritional requirement due to illness, voluntary or forced fasting, and/or starvation. With the increased demand for glucose required for lactose production, relatively few metabolic possibilities exist in the fasting condition: 1) the lactating mothers must produce more glucose than the nonlactating women; 2) the mothers must reduce their own body’s use of glucose via increased availability and use of FFA and ketone bodies; 3) the mothers will develop hypoglycemia; and/or 4) mothers will decrease lactose synthesis and, thus, milk production. On the basis of the propensity of adult women to relative fasting hypoglycemia and hyperketonemia when compared with men (4, 5, 6), we hypothesized that the primary mechanism for maternal adaptation to short-term fasting is decreased maternal glucose use by the early development of ketosis and fatty acidemia, thus maintaining a glucose supply for milk production and a constant milk supply for the infant. Furthermore, we would hypothesize that in the fed state, glucose production in lactating women would be incompletely suppressed.
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Materials and Methods |
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[U-13C]glucose (99+ atom % 13C), [1,1,2,3,3-2H5]glycerol (98+ atom % 2H), [2-13C]glycerol (99 atom % 13C), [6,6-2H2]glucose (99 atom % 2H) and [1-13C]glucose (99 atom % 13C) were obtained from Cambridge Isotope Laboratories (Andover, MA). The isotopes were dissolved in 0.45% saline, and the solution was filtered through a 0.2-µm Millipore Corp. (Bedford, MA) filter into sterile syringes. Sterile isotope solutions were prepared less than 48 h before study and maintained at 4 C until used. Isotope solutions were tested and found to be sterile and pyrogen free.
Study design
Subjects.
The study was approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine and the General Clinical Research Center (GCRC) Advisory Committee at Texas Children’s Hospital (Houston, TX). Written consent was obtained from each of the subjects. Fourteen (six and eight in protocols 1 and 2, respectively) normal lactating women and their infants were recruited for these studies (see below). The women were between 18–35 yr of age, and between 6 wk and 3 months postpartum (Table 1
). The term infants of these women were healthy and exclusively breast-fed at the time of the study. Twelve (six in each of protocols 1 and 2) healthy nonpregnant, age-matched, nonlactating women were recruited as controls (Table 1). All volunteers had a normal physical examination, normal hemoglobin and screening studies for liver and renal function, and a negative pregnancy test before they were accepted into the study.
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In protocol 1, we attempted to maintain energy neutrality and used a commercially available preparation containing unlabeled complex carbohydrate. As a result, we were unable to estimate glucose splanchnic extraction and endogenous glucose production. Therefore, protocol 2 was designed to address these issues.
Protocol 1: Sustacal feeding and short-term fasting. Each woman and her infant were admitted to the Metabolic Research Unit at the Children’s Nutrition Research Center (CNRC) or the GCRC on the evening before study. At 1800 h on the evening of admission, two iv catheters were introduced in the antecubital fossae or forearm veins under EMLA (Astra Pharmaceuticals, Wayne, PA) cream analgesia, one for isotope infusion and the other in the contralateral arm for blood sampling. Subjects were fed a supper meal of 10 kcal/kg at 1800 h and subsequently fasted except for water overnight. At 0600 h, baseline breast milk (lactating women) and blood samples (5 ml each) were obtained, and the lactating women received an oral dose of 2H2O (0.1 mg/kg) to measure the equilibration of body and milk water and to determine the turnover of the breast milk water and presumably milk lactose. These data are presented in the accompanying manuscript (3).
Beginning at 0600 h, all subjects received a primed-constant infusion of [2-13C]glycerol (30 µmol/kg; 2.0 µmol·kg-1·min-1 during feeding and 1.5 µmol·kg-1·min-1 during the fasting study) to measure both glycerol kinetics and the fraction of glucose derived from gluconeogenesis. In the initial three women in each group, [2-13C]glycerol was infused at 1.0 µmol·kg-1·min-1 during feeding, resulting in too low enrichment in the M+2 glucose isotopomers for accurate measurement of gluconeogenesis; therefore, the last three women studied in each group were infused at 2.0 µmol/kg-1-min-1, and the data for gluconeogenesis in the fed state are based on these three women in each group. All other results represent the data from six lactating women and six controls, respectively. In addition, [U-13C]glucose (20 µmol/kg; 0.33 µmol·kg-1·min-1) was infused beginning at 0600 h to measure total glucose rate of appearance (Ra). Blood samples (6–10 ml) were collected at 0600, 0900, 1200, 1445, 1500, 1515, 1530, 1545, 1600, 1630, and 1800 h. The infants were fed from both breasts at approximately 3-h intervals, and milk samples were obtained before and after each feeding. To determine the volume of milk consumed, infants were weighed before and after each breast-feeding (7). The volume of milk produced was the sum of the milk consumed and that collected for analysis. Nonlactating women were studied using an identical protocol, except that 2H2O was not administered and no breast milk samples were obviously obtained.
Each woman in both groups was studied on two occasions: once with continuous feedings and once during a brief fast. For the fed protocol, the nonlactating women received approximately 67 kcal (
67 ml) of a nonlactose-containing nutrient drink (Sustacal; Mead Johnson Nutritionals, Evansville, IN) every 30 min from 0600–1800 h. This provided approximately 1600 kcal over the period of study. Lactating women drank 90 kcal (90 ml) every 30 min between 0600–1800 h, providing approximately 2160 kcal. The difference in the caloric intake between the lactating and nonlactating women reflects the differences in the measured daily caloric intake between lactating and nonlactating women (8, 9, 10) and accounts for the estimated calories lost in breast milk over a 24-h period (overnight plus 12-h study). During the fasting study, the women drank an equal volume of water but received no calories from 1800 h the night before study to 1800 h on the day of study. Each study was separated by 2–4 wk, and the fed and fasted protocols were carried out in random order.
Protocol 2: Identical oral glucose load.
Each woman was studied on one occasion using a protocol identical to that outlined above except that 1) the only isotope infused during the study was [6,6-2H2]glucose (20 µml/kg; 0.33 µmol·kg-1·min-1) to measure the total rate of glucose entering the systemic circulation; 2) they received only oral glucose enriched (2.5%) with [1-13C]glucose every 30 min at 33 µmol·kg-1·min·1 to measure the rate of dietary glucose entering the systemic circulation (i.e. no 2H2O, [U-13C]glucose, or [2-13C]glycerol was administered); 3) breath samples were collected every 3 h, and rate of CO2 expired (
CO2) was determined using a MedGraphic Model Indirect Calorimeter (MedGraphics Inc., Minneapolis, MN); and 4) the study duration was 9 h.
Analyses. Plasma glucose was measured using an enzyme-specific method (YSI, Inc. Glucose Analyzer, Yellow Springs, OH). Plasma insulin and C-peptide were measured using commercially available RIA kits (Linco Research, Inc., St. Charles, MO). Plasma FFA and ß- hydroxybutyrate were determined by microfluorometric enzyme analyses using a Cobas Fara II Analyzer (Roche Diagnostic Systems, Inc., Montclair, NJ). Plasma glycerol concentrations were determined by reverse isotope dilution and gas chromatography-mass spectrometry (GCMS) using [2H5]glycerol as an internal standard (11, 12).
The penta-acetate derivative of glucose and the triacetate derivative of glycerol were prepared as described previously (13, 14). The isotopic enrichments of [6,6-2H2]glucose were measured by GCMS using a quadrupole instrument (HP 5890/HP5970; Hewlett-Packard Co., Palo Alto, CA) and an HP-1701 column (30 m x 0.25 mm x 0.25 µm) (Agilent Technologies, Wilmington, DE). Electron impact ionization mode was used with selected ion monitoring of m/z 242–244. The 13C isotopic enrichments in plasma glucose and in the glucose drinks provided were measured by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS): GC, HP 5890 (with a HP1701 column, 30 m x 0.25 mm x 1 µm); C, Europa, Orchid; IRMS, Europa 2020 (Europa Scientific, Crewe, UK). Enrichments of 13CO2 in breath samples were measured by IRMS using Europa, Roboprep G and Tracermass (Europa Scientific). Enrichments of [2-13C]glycerol, [2H5]glycerol, [U-13C]glucose, and the glucose isotopomers were measured by GCMS using a quadrupole instrument (HP 6890/HP5973, Hewlett-Packard Co.) and an HP-1701 column (30 m x 0.25 mm x 0.25 µm) (Agilent Technologies). Positive chemical ionization mode was used with methane as the reagent gas and selected ion monitoring of m/z 331–337 for glucose, and m/z 159, 160, and 164 for glycerol. All measurements were made in the Stable Isotope Core Laboratory of the CNRC.
Calculations.
The Ra of glucose and glycerol into the systemic circulation and the GPR during fasting were calculated under near steady-state condition using standard isotope dilution equations:
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The fraction of glucose production derived from gluconeogenesis and the rate of gluconeogenesis were estimated using mass isotopomer distribution analysis as described by Hellerstein et al. (12, 15, 16). In the fasting state, rates of glycogenolysis were determined by subtracting the rate of gluconeogenesis from the total glucose Ra.
We assume that the enterally delivered glucose ingested in protocol 2 was completely absorbed. The rate of entry of the dietary glucose into the systemic circulation was calculated tracing the Ra of the enterally ingested tracer ([1-13C]glucose) using the infused tracer ([6,6-2 H2] glucose) and correcting for the enrichment of the ingested tracer in the oral glucose using the following equation (17):
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The fraction of dietary glucose entering the systemic circulation was calculated as the ratio between the enteral glucose Rasystemic and the rate of glucose ingestion.
The rate of glucose production (GPR) during ingestion of the glucose drink was calculated using the following equation:
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CO2 is the rate of CO2 expired (µmol·kg-1·min-1), [1-13C]glucoseplasma is the enrichment of [1-13C]glucose in the systemic circulation, and 0.8 corrects for recovery of labeled CO2.
The rate of glucose storage was calculated using the following equation:
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Glucose is the plasma glucose contribution to the rate of lactose synthesis (estimated at the rate of 80% of the lactose production in the lactating women) (3). Statistical analysis. Data within a group were compared using a paired t test. Data between groups were compared using a nonpaired t test. In Protocol 1, in both the fed and the fasting condition, the baseline substrate and hormone values from the overnight 12-h fasts (0600 h) were averaged for each individual and compared with those obtained between 1615–1800 h, i.e. after 22.25–24 h of fasting or 10.25–12 h of feeding. The glucose kinetic data, (glucose Ra, GPRs, and gluconeogenesis) represent the average of the values measured between 1615–1800 h, i.e. after 22.25–24 h of fasting or 10.25–12 h of feeding. In protocol 2, substrate and hormone data from the overnight 12-h fast (baseline, 0600 h) were compared with the values measured from 1245–1500 h, i.e. after 6.75–9 h of glucose ingestion. The glucose kinetic data (glucose Ra, glucose production, splanchnic extraction of dietary glucose, oxidation and storage rates) represent the average of the values measured between 1245–1500 h, i.e. after 6.75–9 h of glucose ingestion. It should be noted that neither the hormone and substrate data nor the glucose kinetic data obtained during the 1 h of frequent sampling (every 15 min) differed from the results obtained during the above mentioned time periods used for statistical analyses (data not shown). All data are expressed as mean ± SE.
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Results |
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Plasma substrate and hormone concentrations.
After a 12-h overnight fast, the plasma concentrations of glucose, lactate, ß-hydroxybutyrate, FFA, insulin, and glucagon were similar in both groups. After 12 additional hours of fasting (24-h total fast), plasma glucose and insulin decreased similarly (P < 0.01) in both groups. Plasma ß-hydroxybutyrate and FFA increased as anticipated, but the increases were similar (P < 0.01) in both groups of women. Plasma lactate and glucagon were similar in both groups and did not change over time in either group with fasting (Table 2). One of the subjects in the control group was excluded from the glucagon analysis, because the values at all time points were more than 10 SD values higher than the group mean.
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). The fractions of glucose derived from gluconeogenesis were similar in the lactating and nonlactating women (0.30 ± 0.03 vs. 0.38 ± 0.03, respectively; P > 0.1). The rates of gluconeogenesis in the lactating and nonlactating women were indistinguishable (3.65 ± 0.34 vs. 3.53 ± 0.34 µmol·kg-1·min-1; P = NS). In contrast, the rates of glycogenolysis were 50% higher (P < 0.05) in the lactating women when compared with the controls (8.86 ± 1.06 vs. 5.85 ± 0.50 µmol·kg-1·min-1) (Fig. 1).
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Milk volumes. During the fast, the milk volume production was 99 ± 9 ml/feeding.
Response to feedings
Substrate and hormone concentrations.
After 10.25–12 h of frequent feeding of Sustacal, plasma substrate and hormone concentrations were essentially the same between the lactating and nonlactating women despite a 30% higher rate of Sustacal intake in the lactating women (Table 2
). Similarly, no differences were observed in the plasma concentrations of substrates and hormones between the two groups of women during ingestion of identical rates of glucose labeled with [1-13C]glucose (Table 3). However, during glucose ingestion, insulin concentrations were lower in the lactating women compared with controls (L, 14.9 ± 3 µU/ml; NL, 28.23 ± 6.0 µU/ml; P = 0.05) (Table 3). Although a similar trend was observed for the C-peptide values, the difference did not reach statistical significance (L, 3.51 ± 0.5 ng/ml; NL, 5.16 ± 0.6 ng/ml; P = 0.08).
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). The fraction of glucose Ra derived from gluconeogenesis was similar in the lactating and nonlactating women (0.16 ± 0.01 vs. 0.16 ± 0.03). Assuming that endogenous glucose production was exclusively from gluconeogenesis, a minimal estimate of GPR during meal ingestion, was similar in both groups (4.79 ± 0.48 vs. 4.61 ± 1.08 µmol·kg-1·min-1 in lactating women and controls, respectively) (P = NS) (Fig. 1). The rates of carbohydrate ingested were intentionally different between the lactating and nonlactating women, and no tracers were included in the complex starches of the feeding (see protocol design). Therefore, we were unable to measure the rate of splanchnic extraction, endogenous glucose production, and the fraction of the dietary glucose entering the systemic circulation. These issues were addressed in protocol 2, below.
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). However, the GPR was slightly, but significantly higher in the lactating women (1.8 ± 0.2 vs. 1.2 ± 0.2 µmol·kg-1·min-1; P < 0.04 by one-tailed t test) (Table 4 and Fig. 2).
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Glucose storage.
The amount of glucose that was neither oxidized nor lost in milk lactose production (in the lactating women) was similar in both groups of women (18.0 ± 0.8 µmol·kg-1·min-1 in the lactating women vs. 20.0 ± 1.5 µmol·kg-1·min-1 in the nonlactating women; P = NS) (Fig. 2).
Glycerol kinetics. During Sustacal ingestion, the Ra of glycerol was similar in the lactating and nonlactating women [2.93 ± 0.30 and 2.56 ± 0.23 µmol·kg-1·min-1, respectively ( P = NS)].
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Discussion |
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This increased glucose demand is not trivial and amounts to more than 50 g/d above that of the fasted controls. We clearly demonstrated that over the period of study the lactating women met their increased glucose demands by increasing their rates of glycogenolysis, and not by 1) increasing their rates of gluconeogenesis or glycerol turnover (indicator of lipolysis), or 2) decreasing glucose use or milk production. There was, however, a great intersubject variation in milk production. Considering the anecdotal histories of lactating women always being hungry and constantly eating, we were reluctant to extend their fast further until more data were available. As a result, we recognize that the fast that we used may have been an inadequate challenge to elucidate clearly the maternal adaptation to caloric restriction. However, it would appear that one potential adaptive mechanism that cannot be proved from this short-term fasting study is the potential of decreasing milk (and thus, lactose) production.
Of equal interest is the potential mechanism(s) by which lactating women meet their increased carbohydrate needs in the fed state. Increased peripheral glucose demands could be met in the fed condition by incomplete suppression of hepatic glucose production, altered hepatic glucose uptake, and/or decreased storage of glucose after a meal. No differences were observed in splanchnic and systemic metabolism of ingested glucose between lactating women and controls. Although a significant difference was observed in the GPRs between the two groups, quantitatively, this is probably not of any physiological importance. Within the limitation of the methods used, it would appear that lactating women handle a glucose load in a fashion that is essentially identical to that of the nonlactating women.
It is of interest to note that in both the lactating and nonlactating women, the rate of gluconeogenesis was at least the same and perhaps slightly increased with feeding. Because the only source of doubly labeled glucose could be from the [2-13C]glycerol via gluconeogenesis, our data demonstrate that even after 12 h of continuous stimulation of enteral feedings, glucose continues to be released into the systemic circulation via the gluconeogenic pathway. This would suggest that during meal ingestion, the rate of glucose appearing from gluconeogenesis is not acutely regulated by insulin but that the primary role of insulin, under these conditions, may be to modulate the rate of systemic glucose derived from glycogen alone.
During ingestion of identical amounts of glucose, plasma glucose concentrations were similar but insulin concentrations were lower in the lactating women compared with controls. In addition, during ingestion of Sustacal, insulin concentrations were similar in the two groups, although by study design the lactating women received approximately 30% more carbohydrate than the nonlactating controls. Thus, on the basis of insulin to glucose ratios, the results of these studies indicate a change in insulin sensitivity during lactation. Human as well as animal studies suggest an insulin-independent mechanism for glucose uptake by the mammary gland; Neville et al. (18) observed no effect on milk volume, milk glucose concentration, total fat content, or lactose secretion rate during a 4-h hyperinsulinemic euglycemic clamp. Hyperglycemia induced by acute withdrawal of insulin infusion did not change lactose levels in goats, (19) although chronic insulin deficiency was associated with a reduction in milk production (20). To explore these issues and determine the potential role of insulin during lactation will require further studies.
In conclusion, the increased glucose demands of lactation are met by increased GPR as a result of increased glycogenolysis but not by gluconeogenesis or increased use of FFA. During feeding, lactating women handle oral carbohydrates normally but have increased insulin sensitivity. Many women increase their body fat mass with each consecutive pregnancy, because they are unable to lose this fat in the postpartum period regardless of breast-feeding or not (21). This progressive increase in body fat is associated with increased risks of diabetes mellitus, hypertension, and macrovascular disease. By providing insight into some aspects of maternal metabolism during lactation, these data might have great importance in establishing sound dietary guidelines for the lactating woman.
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Acknowledgments |
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Footnotes |
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This work is a publication of the USDA/Agricultural Research Service, CNRC, Department of Pediatrics, Baylor College of Medicine, Houston, TX. The contents of this publication do not necessarily reflect the views of policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement from the U.S. Government.
Abbreviations: CNRC, Children’s Nutrition Research Center; GCMS, gas chromatography-mass spectrometry; GCRC, General Clinical Research Center; GNG, gluconeogenesis; GPR, glucose production rate; L, lactating; NL, nonlactating; Ra, rate of appearance; USDA, U.S. Department of Agriculture; VCO2, rate of CO2 expired.
Received July 13, 2001.
Accepted October 10, 2001.
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