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APPETITE, OBESITY, DIGESTION, AND METABOLISM
1Clore Laboratory, University of Buckingham, Buckingham, United Kingdom; and 2Aberdeen Centre for Energy Regulation and Obesity, School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom
Submitted 26 September 2006 ; accepted in final form 15 January 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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additional energy balance; energy expenditure
Plasma leptin concentrations in both the dam and the developing offspring may play a role in linking nutrition to development. Thus the absence of leptin in Lepob/Lepob mice and their dams results in permanent disruption of hypothalamic development (9). Moreover, we previously showed that administration of leptin from day 14 of pregnancy to rats fed on a low-protein diet totally prevented diet-induced weight gain in their male offspring. Furthermore, these offspring did not respond to the high-fat diet with elevated insulin levels, either in the fasted state or following a glucose load (36).
Here, we have investigated whether administration of leptin to pregnant rats reduces susceptibility to diabetes and obesity in their offspring when the dams receive a normal diet. We have studied whether reduced energy intake or increased expenditure is involved in resistance to obesity. Moreover, because a number of studies have found sex differences in the susceptibility of rodents to obesity and diabetes (11, 17, 19, 22, 37), we have studied female, as well as male offspring. The results presented are for female and male offspring that were challenged with different high-fat diets. These elicited a similar level of obesity in the two sexes, whereas when the same diet was used, obesity was more pronounced in the females.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Pregnant Wistar rats (Charles River, Margate, UK) (initial weight 200–225 g) were received time-mated at day 1 of gestation (taken as when vaginal plugs were detected), housed individually, and maintained at 22°C on a 12:12-h light-dark cycle. They were fed on a diet containing 20% (wt/wt) protein that contained 14.0 kJ/g metabolizable energy throughout pregnancy and lactation. From day 14 of pregnancy, they were given either saline or rat leptin (2 mg·kg–1·day–1 in physiological saline; PeproTech EC, London, UK) via a subcutaneously implanted Alzet minipump (Charles River) for 28 days. Spontaneous delivery took place on day 22 of pregnancy after which, at 2 days old, litter sizes were standardized to nine for each mother. All maternal measures and pup measurements postweaning were taken in the fed state at 1000, with plasma concentrations being measured from tail blood samples. At 21 days of age, all of the pups were housed in groups of three or four and weaned onto the 20% (wt/wt) protein diet until 6 wk of age when half of the female (n = 6 to 8 per treatment group) and one-third (n = 6 to 8) of the male pups were transferred to a high-energy (HE) diet (19.26 kJ/g metabolizable energy) that contained by energy 45% fat, 35% carbohydrate (of which half was sucrose), and 20% protein (Research Diets, D12451 [GenBank] ; New Brunswick, NJ). Another one-third of the male pups were transferred to a high-fat (HF) diet (UAR, Villemoisson, France) that contained by energy 20% protein, 12% carbohydrate, and 68% fat. Results for male offspring fed on the HE diet are not presented because offspring of the leptin-treated dams did not become significantly obese. Throughout the study, all of the rats were allowed to eat ad libitum and had free access to drinking water.
Milk was obtained on day 5 of lactation by milking the dam following separation from the offspring and administration of oxytocin. Samples of milk were weighed to obtain the wet mass and then dried to constant weight (14 days) at 60°C. Energy contents of the solids were then determined on three replicates of the pooled samples by adiabatic microbomb calorimetry (Gallenkamp, Leicestershire, UK).
Energy balance. The daily food intake of the dams was measured during pregnancy and lactation, excluding the days immediately prior to parturition. The food intake of the offspring was measured on three consecutive days over 24 h when they were 3, 5, 9, 12, and 15 mo old.
The body composition of the pups (anesthetized using halothane and nitrous oxide) was measured at 3 to 4 wk of age using dual-energy X-ray absorptiometry (Lunar PIXImus 2 mouse densitometer and version 1.46 software; GE Medical Bedford, UK).
Energy expenditure was measured in offspring by open-circuit indirect calorimetry beginning at 1030 to 1100 for 24 h, or over 6 h following administration of the 
3-adrenergic agonist BRL-37344 (1 mg/ kg body wt ip) (4). The respiratory chambers were the rats' home cages covered with a Perspex lid that had 8-mm holes at each end for entry and exit of air. The system used 12 chambers. Air was sucked through each box by a pump (model C5BS; Gast Manufacturing, High Wycombe, UK), dried with silica gel, passed to a mass flow meter (EZ-FLOW mass flowmeter; Bronkhorst, Cambridge, UK), then to a flow regulator (Fine Control Needle Valve FCVSL; Roxspur Measurement and Control, Sheffield, UK). Each air line was then passed to a sampling unit (Instrument Design Technologies, GlaxoSmithKline, Harlow, UK), which sampled air from a different line every 90 s. Air was pumped from the flow selector via another flow regulator to the oxygen analyzer (Servomex 1440; Servomex Group, Crowborough, UK), and at the end of this time, the percentage oxygen content was measured. A T-junction between the flow selector and the pump to the oxygen analyzer allowed air to escape via a flow indicator (gas flow meter Platon 0–1000 cm3/min; Roxspur Measurement and Control, Sheffield, UK) to ensure that there was a positive pressure between the flow selector and the pump and that room air was not drawn into the system. The volume of the cages was 23 liters, and the flow rate was 0.8 l/min. Such a system has a calculated half-life for responding to a step change in energy expenditure of 23.5 min. Therefore, it is not suitable for instant measurement of energy expenditure, but with the animals undisturbed in their home cages, it is ideal for measurement of 24-h energy expenditure (2). Energy expenditure was calculated by customized software using the equation of Weir (see Ref. 3).
Glucose tolerance and plasma analytes. Intraperitoneal glucose (1 g/kg body wt) tolerance tests were conducted after fasting overnight. Blood samples were taken from the tail for glucose and insulin measurements. Leptin was measured in 3-mo-old offspring after an overnight fast, as previously described (36), and tri-iodothyronine (T3) and thyroid-stimulating hormone (TSH) were measured using kits from GE Healthcare (Amersham, Little Chalfont, Buckinghamshire, UK). The experiment was terminated, and fat-pad weights were measured in fed animals when the offspring were 15 mo old.
Brown adipose tissue mitochondrial protein and uncoupling protein-1. For measurement of mitochondrial protein concentration, a mitochondrial-enriched fraction was isolated from interscapular brown adipose tissue of the male mice using a mitochondrial isolation kit (Sigma Aldrich). Briefly, tissue was homogenized in an ice-cold isotonic extraction buffer containing 10 mM HEPES, pH 7.5, 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 2 mg/ml delipidated BSA that removes free fatty acids. Following a series of low- and high-speed centrifugation steps, the mitochondrial pellet was suspended in 10 mM HEPES, pH 7.4, containing 250 mM sucrose, 1 mM ATP, 0.08 mM ADP, 5 mM sodium succinate, 2 mM K2HPO4, and 1 mM dithiothreitol. The samples were then assayed for their protein concentration using Bradford reagent (Sigma Aldrich, Poole, UK).
For Western blot analysis, protein was extracted from frozen interscapular brown adipose tissue of the male mice in ice-cold RIPA buffer (1% octylphenoxypolyethoxyethanol, 1.5% sodium deoxycholate, 0.1% SDS in PBS) containing protease inhibitors. The protein concentration of the lysate was determined using Bradford reagent. Protein lysates (20–50 µg of protein) were resolved by electrophoresis using Tris·HCl gels in Tris/glycine SDS buffer. Proteins were transferred onto a polyvinylidene fluoride membrane overnight at 4°C in tris-glycine buffer. Equal protein loading and transfer were visualized by staining the membrane with Ponceau stain. The blot was blocked for a minimum of 1 h with 5% Marvel-milk in TBS Tween. Membranes were then incubated with a 0.1 µg/ml rabbit anti-mouse uncoupling protein-1 monoclonal antibody (Chemicon Europe, Eastleigh, UK). A horseradish peroxidase-conjugated secondary antibody (anti-rabbit) was used to bind to the primary antibody, and peroxidase was detected using an ECL kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol. The fluorescence was captured onto photographic film (Kodak Biomax Light Film), and band density was determined using Alpha Innotec software.
Statistics. Glucose tolerance, plasma analyte levels, and pancreatic hormone measurements were analyzed using one-way ANOVA followed by Bonferroni's posttest for selected comparisons, except for some data in Fig. 1, in which variances were significantly different by Bartlett's test, and so the Kruskal-Wallis nonparametric and Dunn's comparison test were used. Regression lines for energy expenditure vs. body weight in 3-mo-old offspring were compared using GraphPad Prism software ver. 3.0, provided that one of the regressions was statistically significant and the slopes of the regressions were not significantly different. Thus the comparison was of energy expenditure for a normalized body weight. The experiment included similar numbers of offspring of dams fed on a low-protein diet, but the data for these animals are not presented because the results are very similar to those previously reported for a different experiment (36). Results are given as means ± SE. Statistical significance for effects of leptin in the figures is given as *P < 0.05; **P < 0.01; ***P < 0.001.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Offspring from birth to the end of the chow feeding period. Litter size was unaffected by treatment with leptin. However, leptin reduced both the birth weight and lengths of the pups (Table 1). Offspring of the leptin-treated dams were lighter than those of saline-treated dams at weaning (data not shown) and at the age of 6 wk (Table 2).
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Weight gain in offspring in response to diets. The HE diet (45% fat by energy) increased weight gain in the female offspring of the dams that had been treated with saline. Administration of leptin to the dams protected the female offspring from the effect of the HE diet on weight gain (Fig. 2A). The HE diet was relatively ineffective in producing weight gain in the male offspring of dams, even of those dams that had been treated with saline, and the data are not shown. The HF diet (68% fat by energy) promoted weight gain and obesity in the male offspring to a similar degree to the HE diet in the female offspring but again only in the offspring of dams that had been treated with saline and not in offspring of dams treated with leptin (Fig. 2B).
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Energy intake in offspring response to diets. At 3 mo of age, there was a trend for energy intake per rat to be higher on the HE diet than on the chow diet in female offspring of the saline-treated but not of the leptin-treated dams (Fig. 3A). This trend had disappeared by 5 mo of age, however, and relative to body weight, the offspring on the HE diet actually consumed less energy at 5 mo of age, irrespective of their mother's treatment (Fig. 3B).
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Energy expenditure in offspring in response to diets. Treatment of the dams had no effect on energy expenditure of the offspring aged 3 or 12 mo when energy expenditure was expressed per rat (Fig. 4A for 3 mo; 12 mo not shown). In general, however, mean energy expenditure relative to body weight was higher in offspring of dams treated with leptin, and this effect was statistically significant in females aged 3 mo and fed on the HE diet (Fig. 4B). Comparison of regression lines for energy expenditure vs. body weight in 3-mo-old offspring supported this conclusion. This regression was significant (r2 = 0.92; n = 6; P < 0.01) for female offspring of saline-treated, but not leptin-treated, dams fed on the HE diet, probably because body weights varied less in the offspring of the leptin-treated dams. All of the values for the offspring of the leptin-treated dams sat above the regression line for the offspring of the leptin-treated dams, such that energy expenditure at the mean body weight of the leptin-treated dams was 15% higher than at the same body weight on the regression line of the saline-treated dams (P = 0.015). A similar situation and a similar trend of increased energy expenditure in offspring of leptin-treated dams were seen in 3-mo-old male offspring fed on the HF diet (11%; n = 6; P = 0.063).
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The HF diet increased the amount of mitochondrial protein in the interscapular fat pad of the male offspring, whether this was expressed per fat pad or relative to fat-pad weight (saline-treated dams: chow, 2.11 ± 0.23; HF diet, 6.21 ± 0.81 µg protein/mg tissue; P < 0.05); leptin-treated dams: chow, 2.25 ± 0.21; HF diet, 7.34 ± 1.37 µg protein/mg tissue; P < 0.01). Treatment of the dams with leptin or saline had no effect on the amount of mitochondrial protein, however. The HF diet also increased (P < 0.001) the uncoupling protein-1 protein concentrations per unit of tissue protein (saline-treated dams: chow, 2,632 ± 174; HF diet, 4,258 ± 370 arbitrary units; P < 0.001; leptin-treated dams: chow, 2,525 ± 109; HF diet, 4,194 ± 207 arbitrary units; P < 0.001), but again leptin had no effect. Plasma T3 and TSH concentrations were unaffected by the treatment of the dams or the diet of the offspring (T3, saline-treated dams: chow, 1.31 ± 0.53; HF diet, 2.01 ± 0.69 ng/ml; leptin-treated dams: chow, 1.21 ± 0.20; HF diet, 1.96 ± 0.73 ng/ml; TSH, saline-treated dams: chow, 14 ± 2; HF diet, 11 ± 2 ng/ml; leptin-treated dams: chow, 13 ± 1; HF diet, 14 ± 1 ng/ml).
Glucose homeostasis in offspring in response to diets. Treatment of the dams with leptin protected their female offspring from impaired glucose tolerance at the age of 14 mo in response to the HE diet (Fig. 5, A and C). The fasting plasma insulin concentration was also less affected by the HE diet in the offspring of the leptin-treated dams (Fig. 5D).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Increased adiposity occurs in the offspring of high-fat-fed rats (5), as well as in the offspring of undernourished dams. Our results do not support the suggestion that obesity in the offspring of high-fat-fed dams is a consequence of elevated plasma leptin concentrations in the dams but do not exclude the possibility that the cause of obesity in these offspring is a premature surge in plasma leptin concentration 8 to 10 days after birth (42).
We have extended the findings of our previous study (36), in which the dams were fed on a low-protein diet and the male offspring of leptin-treated dams were found to be resistant to high-fat diet-induced obesity. In the present study, the female offspring were fed on a high-energy diet that contains a lower proportion of energy in the form of fat than did the high-fat diet. The male offspring were given either the high-fat or the high-energy diet, but the high-energy diet was ineffective in producing obesity, whereas the high-fat diet elicited a similar degree of obesity to that elicited by the high-energy diet in the females. Therefore, only data for the high-fat diet are presented. The difference in diets as well as in sex may explain why treatment of the dams with leptin was more effective in female than male offspring in preventing diet-induced increases in retroperitoneal fat-pad weights, plasma leptin, fasting plasma insulin, and glucose intolerance, although enhanced weight gain was prevented in both sexes.
The reason for feeding the dams on a low-protein diet in our previous study (36) was that we hypothesized that this diet would lower the plasma leptin level and that low exposure of the fetus to leptin might sensitize it to diet-induced obesity. Thus, there are other examples of the consequences of leanness being prevented by administration of leptin, although leanness itself is not prevented (1). In our recent unpublished work, we have found, as we originally predicted, a reduced plasma leptin concentration in dams fed on the low-protein diet, but in the published study (36), the plasma leptin concentration was not detectably low in these dams, suggesting that leptin might be able to benefit the offspring by altering normal development. This was confirmed in the present study.
Our results are in some respects similar to those of Nilsson et al. (28). They reported that injections of leptin into pregnant rats on days 8, 10, and 12 of gestation reduced adipose tissue weight in 10-wk-old male and 12-wk-old female offspring. Body weight was also reduced in the adult females. The offspring in these experiments were all fed on a low-fat diet, however. We did not detect any significant differences in fat- pad weights or plasma leptin levels at 15 mo of age when the offspring were fed on chow: it was only when they were fed on the HE or HF diets that such differences were apparent. Nilsson et al. also reported that their offspring showed reduced skeletal growth, and the females, but not the males, had a reduced body weight. This is consistent with the reduced birth length in our mixed-sex offspring and reduced body weight in the female, but not the male, offspring fed on the chow diet.
An unresolved question is whether leptin administered to the dam reaches the fetus and directly affects its development, or whether its effect is indirect. It might be argued that leptin's effect was an indirect consequence of its negative effect on energy balance in the dams, but the effect on energy balance was small despite the high-leptin dose, suggesting that energy balance is resistant to leptin not only during pregnancy (27) but also during lactation (16). We previously presented evidence that leptin might reduce exposure of the fetus to corticosterone by increasing the activity of placental 11-hydroxysteroid dehydrogenase type 2. However, leptin crosses the placenta, especially during late gestation in rats and might also affect the fetus directly (35). We have found (unpublished data) an elevated plasma leptin level in fetuses of dams that were injected with leptin on day 18 of pregnancy. Moreover, transplacental passage of leptin is decreased by glucocorticoids (35), and so the increased activity of 11-hydroxysteroid dehydrogenase type 2 might increase exposure of the fetus to leptin, as well as decreasing exposure to corticosterone. Recent reports have highlighted direct effects of leptin on hypothalamic development. Specifically, the absence of leptin in Lepob/Lepob mice disrupts the development of neuronal projection pathways from the arcuate to other hypothalamic nuclei (8, 9). Treatment of neonates with leptin rescued the development of these pathways (9). Our study raises the possibility that administration of leptin to pregnant rats permanently enhances the development of fetal neuronal projection pathways from the arcuate to other hypothalamic nuclei in a manner that protects the offspring from dietary obesity. Whether leptin acted directly via hypothalamic leptin receptors in Lepob/Lepob mice (9), or in the offspring of our experiments has not been established.
Leptin given to dams solely during gestation can alter body composition in offspring (28), and we showed that administration of leptin to lactating rats did not affect the energy content of their milk. However, because treatment of neonates rescued the development of neuronal projection pathways in Lepob/Lepob mice (9), it is feasible that the markedly increased concentration of leptin in the milk of leptin-treated dams played a role in our study. This leptin can, to some degree, pass into the infant's circulation (12, 25, 33), and it has been suggested that leptin in breast milk protects children from subsequent obesity (26). It does not support this hypothesis that administration of leptin to lactating, normally nourished rats increased weight gain and adiposity in their offspring (24). Leptin was administered to the dams only during the last 3 days of lactation in this previous study, however, and it may be that the pattern of variation in plasma leptin concentration in suckling rodents is critical (34, 42). There are contrasting reports of the effects of administering leptin to the neonates themselves. In one study, injection of leptin into neonatal rats increased their food intake and body weight when they became adults (15); this effect being more pronounced when leptin was given during the first rather than the last 10 days of suckling. Moreover, administration of leptin to suckling mice from day 5.5 to 10.5 increased their sensitivity to a high-fat diet given from 9 wk of age (42). By contrast, in another study, administration of leptin to neonatal offspring of undernourished rats protected them from later diet-induced obesity (40). This latter study, is consistent with the view that it was the presence of leptin in milk that was critical in our study, except that there was no demonstrable effect of leptin in offspring of normally fed dams (40), whereas the protective effect of leptin in our current study was achieved in dams that were fed normally. These comparisons are, of course, confounded by the fact that administration of leptin directly to the offspring may produce far higher concentrations of leptin in the offspring than was achieved in the present study through its transfer in milk.
We addressed the question of whether treatment of the dams with leptin protected their offspring from diet-induced obesity by reducing energy intake or increasing energy expenditure. Interpretation of such data is complicated if body weights differ between treatment groups, because larger animals are expected to have higher energy intakes and expenditures. Moreover, because intake and expenditure are not directly proportional to body weight, expression of energy turnover relative to body weight is not ideal. A better approach is to compare the regression lines of energy intake or expenditure vs. body weight between groups, but this approach depends on these regressions being significant, which is more likely when body weights vary widely (3).
Treatment of the dams with leptin prevented the male offspring from increasing their energy intake (expressed per rat) on the HF diet. Although the increase in energy intake in offspring of saline-treated dams fed on the HF diet could have been secondary to increased body weight, it is notable that even when energy intake was divided by body weight—an overcorrection because the extra body weight contained a high proportion of fat—energy intake at 9 mo of age was increased by the HF diet in the male offspring of the saline-treated but not the leptin-treated dams.
Energy expenditure per rat was not affected by treatment. Relative to body weight, energy expenditure was higher in females fed on the HE diet if their mothers had been treated with leptin. This may, however, reflect a greater proportion of metabolically active lean tissue in the offspring of the leptin-treated dams. It is more convincing that regression analysis showed that, for the same body weight, energy expenditure was higher in the female offspring of the leptin-treated compared with the saline-treated dams. A similar trend was seen in the males fed on the HF diet. Thus, administration of leptin to the dams appears to protect their offspring from dietary obesity by influencing either or both of energy intake and energy expenditure. This is unsurprising because many peptides that regulate energy balance influence both energy intake and expenditure in a synergistic manner.
We investigated whether there might be an increased capacity for thermogenesis in brown adipose tissue in offspring of leptin-treated dams, but found no effect of leptin on the mitochondrial protein and uncoupling protein-1 content of the interscapular fat pad. Increases would have been expected if sympathetic activity had been increased. Consistent with these findings, there was no significant difference in the thermogenic effect of the 3-adrenoceptor agonist BRL-37344 between the HE diet-fed female offspring of saline- and leptin-treated dams. Possible mechanisms for effects of leptin treatment on energy expenditure suggested by other work include increased testosterone levels (28) and thyroid function (14, 38), but we found no effect of leptin on plasma T3 or TSH concentrations in male offspring.
In conclusion, administration of leptin to pregnant rats during the third trimester of pregnancy and lactation reduced susceptibility to diet-induced obesity, glucose intolerance, and insulin resistance in their offspring, especially the females. Leptin programmed both reduced intake of the obesogenic diets and increased energy expenditure. These results show that leptin levels in dams can affect the development of energy balance regulatory systems in their offspring.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-adrenoceptor on brown adipocytes as target for anti-obesity drugs. Nature 309: 163–165, 1984.[CrossRef][Medline]This article has been cited by other articles:
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  J. Ferezou-Viala, A.-F. Roy, C. Serougne, D. Gripois, M. Parquet, V. Bailleux, A. Gertler, B. Delplanque, J. Djiane, M. Riottot, et al. Long-term consequences of maternal high-fat feeding on hypothalamic leptin sensitivity and diet-induced obesity in the offspring Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1056 - R1062. [Abstract] [Full Text] [PDF]
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 G. Watts In search of fat profits BMJ, June 23, 2007; 334(7607): 1298 - 1299. [Full Text] [PDF]
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) and leptin-treated (
) dams. Results are means of 6 to 9 values with bars for SE. A: plasma leptin levels were significantly (P < 0.05) raised at all times in the leptin-infused rats (one-way ANOVA and Bonferroni's post test, except at times where variances were significantly different by Bartlett's test, in which case the Kruskal-Wallis nonparametric and Dunn's comparison test were used). B: leptin reduced food intake on days 15 and 17 of pregnancy. Repeated-measures ANOVA suggested (P = 0.05) an effect of leptin on days 5 to 17 of lactation (5L and 17L). C: leptin reduced body weight significantly on days 7L and 27L. *P < 0.05; **P < 0.01; ***P < 0.001 for effects of leptin.
, saline; 
, leptin. C and D: saline and leptin refer to treatment of the dams, and female and male to the sex of the offspring. *P < 0.05; **P < 0.01; ***P < 0.001 for effects of leptin.
