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Laboratorio de Biología Molecular, Nutrición y Biotecnología (Nutrigenómica), Universidad de las Islas Baleares, 07122 Palma de Mallorca, Spain; and Centro de Investigación Biomédica en Red Fisiopatología obesidad y nutrición (CB06/03), Instituto Salud Carlos III, Spain
Address all correspondence and requests for reprints to: Andreu Palou, University of the Balearic Islands, Cra Valldemossa, km 7.5, 07122 Palma de Mallorca, Spain. E-mail: andreu.palou{at}uib.es and cati.pico{at}uib.es.
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Abstract |
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Introduction |
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During the early period of life, the brain is particularly sensitive to external factors such as stress and nutritional conditions (7). Indeed, several studies have pointed out the importance of such factors for the regulation of body weight later in life; this has led to the hypothesis of fetal origins of obesity and other related diseases (8, 9). Both prenatal undernutrition and postnatal overnutrition could influence the development of overweight during adulthood (10, 11). What specific food components and the molecular mechanisms underpinning the long-term programming of adult disease, including obesity, are key questions to be faced.
Several studies have shown that breast-feeding, compared with formula feeding, is associated with a lower risk of later obesity (12, 13, 14, 15, 16). Milk is known to contain many bioactive hormones and peptides (17, 18), which may play important roles in neonatal development, and could be responsible for these "programming" mechanisms during these critical periods of development. Leptin is an anorexigenic hormone present in maternal milk (19, 20), but not in infant formula (21), and levels in milk are correlated with maternal body mass index or adiposity and plasma leptin concentration (20, 22). Of interest, we have previously described, in a group of nonobese mothers, the existence of a negative correlation between milk leptin concentration at 1-month lactation and infant body mass index until the age of 24 months (22). Thus, leptin is one of the bioactive components present in milk that could be responsible for the role of breast-feeding in lowering the risk of childhood obesity.
It has been reported that leptin supplied by milk, or leptin supplied as a water solution, can be absorbed by the immature stomach of suckling rats (19, 23, 24) and be transferred to the bloodstream (19, 24). In fact, leptin supplied from maternal milk appears to be the main source of leptin in the stomach during the suckling period, particularly during the first half of this period, in which gastric leptin production is kept low (23). In addition, the administration of physiological oral doses of leptin during the suckling period has inhibited food intake but without affecting body weight gain during this period (24). These results appear to be different from those of Schmidt et al. (25), showing that daily sc administration of pharmacological doses of leptin to rats during the suckling period resulted in lower body fat from d 7 of life onwards. Other authors have found that ip or intracerebroventricular administration of leptin to suckling mice failed to influence milk intake and body weight (26, 27). Differences between the results of these studies could be attributed, at least in part, to the different ways of leptin administration and also to the different doses of leptin used.
To ascertain the role of leptin during lactation, we performed a previous study in rats, and we showed that neonate rats that were orally treated with physiological amounts of leptin during the suckling period were more resistant to age-related body weight increase and diet-induced overweight in adulthood (28); these results suggested that leptin treatment during lactation may affect the early programing mechanisms in the leptin signaling system, resulting in adaptive changes in the control of food intake that help to better regulate energy balance in adulthood (28). Results obtained with leptin-deficient (Lepob/Lepob) mice have demonstrated that leptin seems to be required during a neonatal critical period for normal postnatal development of hypothalamic pathways in the arcuate nucleus, which are involved in leptin signal (29). Moreover, recent feedings have also shown that leptin during the neonatal period may also affect hippocampal function in the long term (30); all in all, these results evidence that leptin may be important in the development of different central nervous system areas during a critical window of development.
In the present study, we have attempted to characterize further the long-term effects of the supplementation of neonate rats with physiological doses of leptin during lactation on glucose and leptin homeostasis and on food preferences.
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Materials and Methods |
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The animal protocol followed in this study was reviewed and approved by the Bioethical Committee of our University, and guidelines for the use and care of laboratory animals of the University were followed.
Study of the adaptations to feeding/fasting conditions
At 9 months of age (3 months after the establishment of significant differences in body weight between both groups of animals), blood samples were collected from the end of the tail of control and leptin-treated animals, without anesthesia, in heparinized containers under three different feeding conditions: 1) ad libitum feeding, during the first 2 h of the beginning of the light cycle in animals provided with free access to chow diet; 2) fasting, during the first 2 h of the beginning of the light cycle in animals deprived of food for 14 h; and 3) pair-fed refeeding, rats fasted for 14 h and allowed a 3-h refeeding period (from 1000–1300 h) with 3.5 g standard chow diet. This amount of food offered to animals was previously checked (in a pilot experiment made 2 wk before with all animals) to be finished during the 3-h period of refeeding. Plasma was obtained by centrifugation of heparinized blood at 2500 g for 10 min.
Plasma leptin concentration was measured using a mouse leptin ELISA kit (R&D Systems, Minneapolis, MN). Plasma insulin concentration was measured using an ELISA kit (DRG Instruments, Marburg, Germany). Blood glucose concentration was measured by Accu-Chek Glucometer (Roche Diagnostics, Barcelona, Spain).
The homeostatic model assessment for insulin resistance (HOMA-IR) was used to assess insulin resistance. It was calculated from fasting insulin and glucose concentration using the formula of Matthews et al. (31): HOMA-IR = fasting glucose (mmol/liter) x fasting insulin (mU/liter)/22.5.
Oral glucose tolerance test (OGTT)
Insulin and glucose responses to an OGTT were also measured to assess insulin sensitivity. The test was performed at the age of 14 months to check the insulin sensitivity in an advanced state of aging. A load of 1- to 1.5-ml glucose (1.5 g/kg body weight) was orally given to the rats using a pipette at 1000 h, after overnight fasting. Blood samples were taken from the tail of animals before glucose load at time zero, and at 30, 60, 120, and 180 min thereafter (32). Plasma glucose and insulin levels were measured as described previously.
Assessment of sensitivity to exogenous leptin–leptin resistance test
The anorexigenic effect of exogenous leptin relative to saline was studied in both control and leptin-treated animals in adulthood, at 7 months of age, 1 month after the establishment of significant differences in body between both groups of animals. Recombinant murine leptin (PeproTech) dissolved in saline was used. Rats from both control and leptin-treated groups were randomly assigned to one of the following groups: the ip leptin group, which received an ip injection of leptin (2 mg/kg body weight, according to Ref. 33), just before lights off at 2000 h; or the vehicle group, which received saline. After injection, they were returned to their home cages and provided with standard diet. Food intake was measured during the next 1, 2, 12, and 24 h after leptin or saline injection.
One week later, the experiment was repeated under the same conditions but reversing the assignment of animals to the ip leptin or vehicle groups.
Two-bottle preference test
Food preferences were assessed by a two-bottle preference test as previously described (7) with slight modifications. The rats had to choose between two bottles containing either a carbohydrate-rich (CR) liquid diet or a fat-rich (FR) liquid diet. The two diets had identical caloric density (2.31 kcal/g) and the following ingredients: for the CR diet, 10 g/100 ml skimmed milk, 40 g/100 ml sucrose, 4 g/100 ml olive oil, and 0.35 g/100 ml xanthan gum (Sigma, Madrid, Spain); and for the FR diet, 10 g/100 ml skimmed milk, 10 g/100 ml sucrose, 17.3 g/100 ml olive oil, and 0.35 g/100 ml xanthan gum. Before the test started, and during a period of 8 d, the rats were habituated to each bottle given individually on alternate days for 1 h, without withdrawing the standard chow diet. The test was started 2 d after the adaptation period. Solid food was withdrawn at the beginning of the light phase. Two bottles containing preweighed quantities of either the CR or FR diet were placed side-by-side 4 h after the beginning of the light cycle for 1 h. The bottles were then reweighed, and the intake of each diet was determined and corrected for spillage. Spillage was estimated by weighing small collection plates placed underneath the spout of the bottles. To control for the effect of the side, half of the rats had the CR bottle on the left and the FR bottle on the right; this was reversed in the other half. The test was performed on 3 different days and at two stages of life, when rats were 9 and 14 months old (i.e. 3 months after the establishment of significant differences in body between both groups of animals and at a more advanced age, respectively).
Statistical analysis
Given that the animals studied were from three different litters, the effect of the litter was simultaneously factored with all data by repeated measures ANOVA. No interactions between the litter and treatment were found across all the data, thus, data were expressed as mean ± SEM of animals from the three different litters (n = 6 animals per group).
Single comparisons between groups were assessed by the Student’s t test. Multiple comparisons were assessed by repeated measures ANOVA, and then the Bonferroni test was used to compare the mean differences between groups. P < 0.05 was the threshold of significance.
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Results |
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) (P < 0.05; interactive effect between leptin treatment and age, repeated measures ANOVA). Differences were significant from d 188 onwards (P < 0.05, Student’s t test). These differences can be explained, at least partially, by a lower food intake (P < 0.05, repeated measures ANOVA) (Fig. 1B). Cumulative food intake during the whole studied period (from d 21–15 months of age) was significantly lower in leptin-treated animals than in their controls (control: 33317 ± 474 kcal, leptin-treated group: 30827 ± 761 kcal; P < 0.05, Student’s t test). Differences concerning daily food intake between both groups of animals were not found in young rats but appeared to be significant from d 84 of life (Student’s t test), and differences were generally maintained during the studied period. However, when daily food intake of animals was referred to their body weight, no significant differences between both groups of animals were found at any age (Fig. 1C).
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). In both groups of animals, the 14-h fasting period induced a significant decrease in glucose levels, and the 3-h pair-fed refeeding after a 14-h fasting period induced a significant increase in circulating glucose levels (P < 0.05, Bonferroni test).
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). The 3-h pair-fed refeeding after a 14-h fasting period induced a significant increase in insulin levels in control animals (P < 0.05, Bonferroni test), but not in leptin-treated animals. The HOMA-IR index was calculated from fasting insulin and glucose concentrations in both groups of rats at 9 and 14 months of age. This value was significantly lower in leptin-treated animals (2.11 ± 0.36) compared with their controls (4.15 ± 0.60) (P < 0.05, Student’s t test) when rats were 9 months old, and slightly lower (2.85 ± 1.06 vs. 4.01 ± 0.96; P = 0.436) when rats were 14 months old.
We also measured glucose and insulin responses to an OGTT (Fig. 3). We found that the glucose area under the curve (AUC) from 0–180 min was significantly lower in leptin-treated animals (962 ± 24 mmol min/liter) compared with their controls (1052 ± 20 mmol min/liter) (P < 0.05, Student’s t test) (Fig. 3A). The insulin responses were similar in both groups of animals (Fig. 3B), and no significant differences were found in the AUC (0–180 min) (values of 46.4 ± 8.5 nmol min/liter in the control group and 43.0 ± 13.5 nmol min/liter in the leptin-treated group).
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). No significant differences were found as an effect of feeding conditions in either group of animals.
Acute ip administration of leptin (2 mg/kg) resulted, after 1 h, in a slight, but not significant, decrease in relative food intake (expressed as g/kg body weight) in both control and leptin-treated animals, which was more pronounced in the leptin-treated group than in control animals (reductions of 34 and 15%, respectively, compared with their controls that were injected saline) (Fig. 4). This anorectic effect was very short lived in the control group. This anorectic effect was very short lived in the control group, because during the second hour after leptin administration relative food intake was identical to its controls, whereas it was 78% in the leptin-treated group. Considering the effect of ip leptin administration during the first 12 h (corresponding to the dark period), the anorectic effect of leptin was significant in leptin-treated animals (P < 0.05, Bonferroni test), but not in the control group. This anoretic effect of ip leptin was still observed in leptin-treated rats by considering a 24-h period after ip leptin administration.
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). Total relative food intake (expressed as grams per kilogram of body weight) consumed during 1 h, considering both FR and CR diets, was similar in terms of energy between control and leptin-treated rats. However, both groups of animals differed in their dietary preferences. At the age of 9 months, control animals showed a significant preference for the FR diet (P < 0.05, Bonferroni test): they ate 34% of the CR diet and 66% of the FR diet. The leptin-treated group did not show any significant preference and consumed similar amounts of both types of diets (53% of CR and 47% of FR). At the age of 14 months, control rats continued showing a preference for the FR (61%) vs. the CR diet (39%), although the threshold of statistical significance was not reached (P = 0.505), whereas leptin-treated animals showed a significant preference for the CR diet (66%) than the FR diet (34%) (P < 0.05, Bonferroni test).
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Discussion |
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Insulin sensitivity in the presence of physiological changes in glucose and insulin concentrations, e.g. after a meal or an OGTT, is important to better understand insulin resistance in a variety of metabolic conditions. In our previous study (28), we found no significant differences (only slight changes) between control and leptin-treated animals concerning glucose and insulin levels, determined at 6 months of age and under ad libitum-feeding conditions. Here, we studied both parameters when rats were 9 months old and under different feeding conditions. Insulin levels were found to be significantly lower in leptin-treated animals than in their untreated controls, both under feeding and fasting conditions, and interestingly, the increase in insulin levels after 3-h pair-fed refeeding after fasting was greater in control animals than in leptin-treated animals. However, glucose levels were not significantly different between both groups of animals. This indicates that control animals must release more insulin than leptin-treated animals to maintain glucose homeostasis, whereas leptin-treated animals seem to be more resistant to the age-associated increase in insulin resistance. In fact, in humans, the earliest or primary determinant of type 2 diabetes mellitus has been described to be increased rather than decreased insulin secretion, especially the first phase, which is characteristic of the prediabetic phase (34).
To ascertain better the effect of leptin treatment during lactation on insulin sensitivity in adulthood, we also used the HOMA-IR and performed an OGTT. The HOMA-IR was used because it is a valuable method that shows a strong relationship with a euglycemic-hyperinsulinemic clamp (35) and, therefore, has been proposed to be used as an alternative method. The HOMA-IR index was calculated from data of fasting insulin and glucose levels, as described by Matthews et al. (31). With such a method, high HOMA-IR scores denote low insulin sensitivity (i.e. insulin resistance). We found that when rats were 9 months old, the HOMA-IR index was significantly lower in leptin-treated animals (49.2% decrease) compared with their respective controls, which is indicative of higher insulin sensitivity. At the age of 14 months, the difference was less marked (28.9% decrease) and did not reach statistical significance.
The OGTT performed when rats were 14 months old also showed that leptin-treated animals were still more sensitive to the effect of insulin. In response to an oral glucose load, the insulin responses were similar in both groups of animals, but the peak of blood glucose 30 min after glucose administration was slightly lower in leptin-treated animals (6.09 ± 0.22 mmol/liter) than in controls (6.74 ± 0.34 mmol/liter) (P = 0.137). In addition, the glucose AUC was significantly lower in leptin-treated animals, thus showing that insulin-stimulated glucose uptake was higher in this group of animals.
Thus, insulin resistance associated with age seems to be decreased in leptin-treated rats compared with controls, as demonstrated by the lower HOMA-IR value, lower insulin release in response to food intake to maintain normoglycemia, and by decreased glucose levels after oral glucose administration. Insulin resistance is known to play a major role in the development of type 2 diabetes (34, 36) and may also be involved in atherogenesis (37, 38), thus improvement of insulin sensitivity is important in the prevention against the metabolic syndrome.
Animals that were treated with leptin during lactation also displayed in adulthood, at 9 months of age, lower leptin concentration in plasma than their untreated controls, under the different feeding conditions studied. This is in accordance with their lower body weight and lower fat mass, as previously described (28). When rats were 6 months old, we already found slightly lower leptin levels under ad libitum-feeding conditions in leptin-treated animals than in their controls, although differences did not reach statistical significance. Age is known to be associated with an increase in leptin levels, which appears to be largely dependent on the increased body weight and fat content (39). This may contribute to the dysregulation of energy balance that occurs with age and involves impairment of the fasting-induced suppression of leptin production, which may be responsible for the age-associated proneness to obesity (40). Leptin treatment during lactation may help to maintain lower leptin levels and could, therefore, help animals be more responsive to adaptive changes in leptin levels. However, neither in control animals nor in leptin-treated animals did we find significant differences in circulating leptin levels as an effect of fasting or refeeding.
The higher sensitivity of leptin-treated animals to leptin was ascertained by studying the anorexigenic effect of exogenous ip leptin. In fact, the effect of exogenous leptin was found only in leptin-treated rat, but not in controls. The lack of a significant effect of exogenous ip leptin in control animals could be attributed to leptin resistance, and most likely to peripheral leptin resistance, associated with age. The time-dependent development of peripheral leptin resistance has been previously described in other rodent models, such as AKR mice under normal or high-fat diet, while these animals retain sensitivity to centrally administrated leptin (41).
Concurrent hyperinsulinemia has played a pivotal role for the development of leptin resistance and, thus, life-long obesity risk (25). Insulin may either directly, or indirectly via its lipogenic action, stimulate adipocyte leptin production and secretion; leptin in turn suppresses insulin secretion by both central actions and direct actions on β-cells via a dual hormonal feedback loop between adipose tissue and the endocrine pancreas, the proposed adipoinsular axis (42). Therefore, increased adiposity and hyperinsulinemia are likely contributors to the rise in plasma leptin concentration and suggest the development of leptin resistance at the level of β-cells (43).
Modulation of feeding behavior, including not only appetite but also food preferences, may also provide a mechanism through which obesity may be programmed (44). Here, we have seen that supplementation with leptin during lactation affects food preferences later on in life. At the age of 9 months, control animals had a clear preference for FR food, whereas no preferences were found in the leptin-treated group. Five months later, the leptin-treated group established a preference for CR food, and control animals continued with a tendency to prefer fat. In humans, the failure to control obesity is generally associated with increased appetite and preference for highly caloric food, in addition to other factors such as reduced physical activity and increased lipogenic metabolism (45). Thus, changes in food preferences in favor of less caloric food could be of interest to prevent obesity, particularly when energy dense foods are widely available as in our developed societies. Other studies have also shown that changes during the perinatal period may influence long-term appetite and food preferences. In particular, exposure to a maternal low-protein diet during fetal life has enhanced fat preferences (46, 47); these animals also exhibited hypertension, altered glucose handling, and higher abdominal fat mass (44). Thus, maternal nutrition and nutrition during critical phases of development may promote changes in the systems involved in controlling appetite and the perception of palatability, and, therefore, affect susceptibility to obesity, although the specific mechanisms involved remain to be clarified further.
In humans, there are clearly differences concerning food preferences, but the genetic background and the effect of early life nutrition, as well as how they interact to confer susceptibility or resistance to obesity, are not known. Improved diagnosis of individuals at risk may allow treatment and preventive measures, including advice during pregnancy.
In summary, we show here that supplementation of neonate rats with physiological doses of leptin during lactation results in effects later on in life. In particular, these animals are programmed for lower body weight and food intake, and appear to be more sensitive to insulin and leptin. Leptin treatment also affects feeding behavior and enhances preference for CR than FR food. Considering that leptin is not present in infant formula and leptin in human breast milk varies widely among lactating mothers, all in all, these findings may have important implications for the prevention of obesity and metabolic syndrome.
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online November 8, 2007
Abbreviations: AUC, Area under the curve; CR, carbohydrate-rich; FR, fat-rich; HOMA-IR, homeostatic model assessment for insulin resistance; OGTT, oral glucose tolerance test.
Received May 11, 2007.
Accepted for publication October 30, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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