Effects of maternal leptin treatment during lactation on the body weight and leptin resistance of adult offspring

We investigate whether leptin treatment to lactating rats affects food intake, body weight and leptin serum concentration and its anorectic effect on their adult offspring. Lactating rats were divided into 2 groups: Lep-single injected with recombinant rat leptin (8 microg/100 g of body weight, daily for the last 3 consecutive days of lactation) and control group (C) that received the same volume of saline. After weaning all pups had free access to the control diet, their body weight and food intake were monitored at each 4 days until 180 days of age, when they were tested for its food intake and response to either leptin (0.5 mg/kg body wt, ip) or saline vehicle. The offspring of the leptin-treated dams gained more weight and had higher food intake from day 37 onward (p<0.05), higher amount of retroperitoneal white adipose tissue (RPWAT) (37%, p<0.05) and higher leptin serum concentration (40%, p<0.05) at 180 days of age compared to control group. The food intake at 2, 4, 6 and 24 h was unaffected after acute injection of leptin in these animals, suggesting resistance to the anorectic effect of leptin. The maternal leptin treatment during lactation makes their adult offspring more susceptible to overweight with resistance to the anorectic effect of leptin.     

Leptin serum concentration,food intake and body weight in rats whose mothers were exposed to malnutrition during lactation

We had shown that adult animals, whose mothers were submitted to protein or energy restriction during lactation, differ from controls in their body weight and thyroid function. The aim of this study was to evaluate, from birth through six months of age, leptin serum concentration, body weight and food intake in animals whose mothers received protein or energy restricted-diet during lactation as follows: control (C)-23% protein; protein-restricted (PR)-8% protein; energy-restricted (ER)-23% protein, in restricted quantity, according to the mean ingestion of the PR group. After weaning (day 21) all pups had free access the control diet. Body weight of pups from PR mothers were always lower than those from controls (p < 0.05), while body weight of pups from ER mothers surpassed that of the C group significantly at 140 days of age. The food intake was lower in both offspring from PR and ER mothers, normalizing on the 32th day in pups from ER mothers and on the 52th day in pups from PR mothers. Leptin serum concentration in both offspring from PR and ER mothers were significantly decreased on the 12th day (p < 0.05) and increased on the 21st day (p < 0.05) compared to control. After weaning there was no differences among the groups. It is possible that changes in leptin concentration during lactation in the offspring of malnourished groups could permanently modify the setpoint for body weight control.     

Prolactin inhibition in lactating rats changes leptin transfer through the milk.

Malnutrition during lactation reduces milk production and changes pup's leptin serum levels. To test prolactin role in this nutritional state, we evaluated whether prolactin suppression during lactation changes serum leptin in dams, its transfer through the milk, and pup's serum leptin. Lactating rats were treated with bromocryptine (1 mg/twice a day, s.c.) or saline three days before sacrifice (days 2-4 or days 19-21). Food intake and body weight were measured until sacrifice (4th and 21st day). Serum prolactin and leptin were determined by radioimmunoassay. Bromocryptine injected dams had lower serum prolactin and milk production as expected. The mothers presented lower food ingestion (day 21: -25%), lower body weight (day 4: -12%; day 21: -10%), higher serum leptin (day 4: +68%), lower milk leptin on the 4th day (11 times) and higher (8 times) on the 21st day. The offspring of bromocryptine-treated mothers presented lower body weight in both periods of lactation and lower serum leptin on the 4th day (-40%) and higher on the 21st day (+37%) of lactation. We suggest that prolactin, through its effect on leptin secretion into the milk, may play an important role in signalizing maternal nutritional status to the pups.     

Maternal leptin treatment during lactation programs the thyroid function of adult rats

Recently, we showed that both maternal malnutrition during lactation and leptin treatment during the neonatal period program thyroid function. In this study we evaluate whether maternal leptin treatment during lactation programs thyroid function of the offspring in the adulthood. The dams were divided into 2 groups: Lep-daily sc single injected with 8 microg/100 g of body weight with recombinant rat leptin during the last 3 days of lactation and control group (C) that received the same volume of saline. The 180 day-old animals received a single i.p. injection of (125)I (2.22x10(4) Bq) and they were killed 2 h after the injection. Triiodothyronine (T3), thyroxine (T4), thyrotropin (TSH) and leptin concentrations were measured by radioimmunoassay. The milk of leptin-treated mothers on the last day of treatment had higher leptin (p<0.05) concentration. The pups of the leptin-treated mothers had at 21 days an unchanged T3, T4 and leptin serum concentrations with higher TSH (p<0.05). The offspring of Lep mothers had at 180 days a higher T3 (p<0.05) with normal thyroid (125)I uptake, T4 and TSH serum concentrations compared to the controls. So, the mother's hyperleptinaemia during lactation programs to a higher T3 serum concentration on the offspring, probably by a higher leptin transfer through the milk.     

Leptin treatment during lactation increases transfer of iodine through the milk

We have previously shown that protein restriction during lactation is associated with changes in iodine secretion into the milk and that a pup's serum leptin concentration was increased at the end of lactation. So, here we evaluate whether leptin treatment during lactation affects iodine transfer through the milk to the pups. Lactating rats were divided into two groups: the leptin (Lep) group, single injected with recombinant rat leptin (8 microg/100g of body weight, daily for 3 consecutive days), and the control (C) group that received the same volume of saline. We studied iodine transfer to the pups through the milk on Days 4, 12 and 21 of lactation. In those days, the dams were separated from their pups for 4 h. Then, the mothers received an injection of 131I (2.22x10(4) Bq ip) and the pups were allowed to nurse for 2 h. The animals were sacrificed 2 h later. Leptin, total serum T3 and total serum T4 concentrations were higher (P<.05) in pups of Lep mothers only on Day 4, suggesting a higher transfer of leptin through the milk at this period, probably with a direct stimulatory effect on thyroid hormone secretion. In other periods, however, even without a detectable increase in a pup's serum leptin concentration, maternal leptin administration increased the pup's thyroid iodine uptake (Day 12, 39%; Day 21, 34%), probably caused by a higher transfer of iodine through the milk, since they had a higher gastric content of 131I on Days 12 (31%) and 21 (128%).     

Absence of anorectic effect to acute peripheral leptin treatment in adult rats whose mothers were malnourished during lactation.

Diets with restricted energy or protein during lactation programs body weight in the adult offspring. We have investigated the hypothesis that protein or energy-restricted diets during lactation alter the feeding response to peripheral leptin treatment of the adult offspring. Five Wistar rats were randomly assigned to one of the following groups on the day that the offspring were born: C, control diet with 23% protein; PR, protein restricted diet with 8% protein; and ER, energy-restricted, receiving the control diet in restricted quantities, which were calculated according to the mean ingestion of the PR group. After weaning (day 21), two animals from each litter (10 pups in each group) were randomly selected and placed together in the cage with free access to water and standard diet until 150 days of age, when they were tested for its response to either leptin (0.5 mg/kg body wt ip) for groups Clep, PRlep and ERlep or saline vehicle for groups Csal, PRsal and ERsal on food intake. In the control groups, food intake was reduced two hours (36%), four hours (41%) and six hours (25%) after leptin treatment. In contrast, no response was observed to leptin treatment in the PRlep and ERlep groups, suggesting leptin resistance. We demonstrated the development of resistance to the anorectic leptin effect and its program in a critical life period associated to nutritional and hormonal factors.     

Leptin injection during lactation alters thyroid function in adult rats.

The aim of this study was to evaluate the effects of hyperleptinemia during the first ten days of life on thyroid function in adulthood. After birth, pups were separated into two groups: L8 - receiving daily injections of recombinant mouse leptin (8 microg/100 g body weight, sc) and control (C) - receiving the same volume of saline. Both groups were treated for the first 10 days of lactation. The animals were sacrificed at 150 days of age, and the blood was collected for leptin, TSH, total triiodothyronine (TT 3 ) and total thyroxin (TT 4 ) serum concentration determinations by radioimmunoassay. The thyroid gland was excised to determine thyroid iodine uptake. Leptin, TT 3 and TT 4 serum concentrations in L8 group were significantly (108 %, 47 % and 32 %; p < 0.05) higher than that of controls. There was no significant difference between the groups related to thyroid iodine uptake and TSH serum concentration. These data suggest that the first half of lactation period is important in determining thyroid function in adulthood, and that it can be programmed by serum leptin concentration.     

Leptin and prolactin, but not corticosterone, modulate body weight and thyroid function in protein-malnourished lactating rats.

To understand the role of hormonal changes in the lower food ingestion and body weight in protein-restricted lactating rats as well as the higher serum T (3), higher deiodination, iodide and T (3) milk transfer, we measured maternal serum prolactin, leptin, TSH and corticosterone, which are hormones that could influence those parameters. After birth, dams were separated into: control-fed with a 23 % protein diet (n = 12) and PR (protein-restricted)-fed with an 8 % protein diet (n = 12). At the 4 (th) and 21 (st) day of lactation, half of the animals in each group were sacrificed. PR dams presented hyperleptinemia (day 4: + 20 %; day 21: + 19 %; p < 0.05) and hypoprolactinemia (day 4: - 85 %; day 21: - 92 %; p < 0.05), which could help explain the lower food consumption and body weight in lactating PR rats since leptin is anorexigenic and prolactin is orexigenic. Also, this hyperleptinemia could contribute for the increase in serum T (3) of PR dams, since leptin stimulates T (3) production, especially acting on deiodinases. Serum corticosterone was not different between PR and C groups, and TSH was lower only at the end of lactation. Thus, we suggest that both leptin and prolactin could play an important role in the body weight and thyroid hormone changes observed in protein-malnourished lactating rats.     

Chronic leptin administration promotes lipid utilization until fat mass is greatly reduced and preserves lean mass of normal female rats

Leptin is a hormone synthesized and secreted from adipose tissue. To study the physiologic effects of chronic leptin treatment, normal adult female Sprague-Dawley rats were injected subcutaneously for 35 days. Twice daily injections (250 microgram/day, b.i.d.) resulted in a significant (P<0.05) decrease in food intake that was maintained for 10 days before gradually returning to control level by day 21. Leptin decreased body weight by a maximum of 12% of the initial body weight on day 22 and remained reduced for the duration of the treatment. After 35 days of treatment, visible peritoneal adipose tissue was not detected. Body composition analysis showed that chronic injection of leptin resulted in a dramatic decrease in fat content (28+/-2 to 4+/-2 g, P<0.05; mean+/-SEM) while the lean content remained unchanged. Rats pair-fed to the leptin-treated group but treated with vehicle had the same body composition (23+/-3 g fat mass) as that measured for the ad libitum fed controls. Using indirect calorimetry we observed that leptin decreased respiratory quotient and thus increased fat oxidation. Leptin also prevented energy expenditure reduction typically associated with food restriction. Leptin treatment for 35 days decreased plasma triglyceride (0.75+/-0.07 to 0.30+/-0.03 mM, P<0.05), free fatty acid (0.56+/-0.06 to 0.32+/-0.04 mM) and insulin (3.2+/-0.5 to 1. 4+/-0.4 ng/ml, P<0.05) concentrations despite the fact that food intake was normalized by day 35. Withdrawal of leptin triggered hyperphagia indicating that leptin biology remained throughout the duration of the chronic treatment. These data suggest that leptin reduces fat mass by initially decreasing appetite and by maintaining enhanced fat utilization even when food intake has returned to that of vehicle-treated control.     

A physiological role of breast milk leptin in body weight control in developing infants

Objective: Leptin, a hormone that regulates food intake and energy metabolism, is present in breast milk. The aim of this study was to determine whether milk leptin concentration is correlated with maternal circulating leptin and BMI and with body weight gain of infants. Research Methods and Procedures: A group of 28 non‐obese women (BMI between 16.3 and 27.3 kg/m²) who breast‐fed their infants for at least 6 months and their infants were studied. Venous blood and milk samples were obtained from mothers at 1, 3, 6, and 9 months of lactation, and leptin concentration was determined. Infant body weight and height were followed until 2 years of age. Results: During the whole lactation period, milk leptin concentration correlated positively with maternal plasma leptin concentration and with maternal BMI. In addition, milk leptin concentration at 1 month of lactation was negatively correlated with infant BMI at 18 and 24 months of age. A better negative correlation was also found between log milk leptin concentration at 1 and at 3 months of lactation and infant BMI from 12 to 24 months of age. Discussion: We concluded that, in a group of non‐obese mothers, infant body weight during the first 2 years may be influenced by milk leptin concentration during the first stages of lactation. Thus, moderate milk‐borne maternal leptin appears to provide moderate protection to infants from an excess of weight gain. These results seem to point out that milk leptin is an important factor that could explain, at least partially, the major risk of obesity of formula‐fed infants with respect to breast‐fed infants.     

Effect of i.c.v. infusion of the alpha-MSH agonist MTII on meal patterns in male rats following nicotine withdrawal

The present study explored the role of endogenous alpha-MSH in the alteration of meal patterns induced by nicotine (NIC) withdrawal. Male Sprague Dawley rats bearing third ventricle cannulas were placed in computerized food intake monitors. On days 1-21, the rats were given 4 mg/kg/day of NIC or saline (SAL) in four equal i.p. doses during the dark period. NIC suppressed (P < 0.05) food intake only during the first week. The normalization of food intake occurred when the reduced meal size of the NIC injected rats was countered by an increase in meal number. Despite the normalization of 24-h food intake, body weight in NIC rats was decreased (P < 0.05) for 21 days. On day 22, the rats were divided into 4 groups (n's = 7-8 each) and injected into the third ventricle with various doses of the alpha-MSH agonist MTII or artificial cerebrospinal fluid (aCSF): SAL + aCSF, SAL + MTII, NIC + aCSF, NIC + MTII. Infusion of MTII (30 ng/rat) suppressed (P < 0.01) dark phase food intake in both groups, but the NIC + MTII group ate (P < 0.05) more than the SAL + MTII group. Meal number during the dark phase was suppressed by MTII, but the NIC + MTII group took significantly more meals that the SAL + MTII group. Infusion of MTII suppressed meal size in SAL and NIC treated rats, but this effect was attenuated in NIC treated rats. All meal parameters normalized by the day after i.c.v. infusion. These data indicate that NIC treatment differentially affects the neural controls of meal number and meal size and attenuates the suppression by MTII of meal number and meal size.     

Influence of feed intake during pregnancy and lactation on fat body reserve mobilisation, plasma leptin and reproductive function of primiparous lactating sows.

From day 23 of pregnancy, 24 gilts received either a medium (M, n = 16) or a high (H, n = 8) level of feeding calculated to meet 115% or 190% of energy for maintenance, respectively. During lactation, all H sows were fed ad libitum (H-AL) whereas M sows were fed either ad libitum (M-AL, n = 8) or were restricted (M-RE, n = 8) to the amount of feed ingested by H-AL sows. Increased feed intake during pregnancy increased live weight, backfat thickness, and estimated body lipid and protein on days 4 and 25 of lactation (P < 0.05). It also resulted in lower feed intake and higher lipid mobilisation during lactation (P < 0.05) without a detrimental influence on milk production. Activities of malic enzyme and glucose-6-phosphate dehydrogenase from neck fat samples were higher in H than M sows on day 4 (P < 0.05). They decreased during lactation in H sows (P < 0.05). Mean diameter of adipocytes decreased during lactation in the 3 groups (P < 0.05) but did not differ between groups on days 4 and 25. Plasma leptin on days 4, 11, 18 and 25 was higher in H than in M sows (P < 0.05) but was not influenced by lactational feed intake. Neither measured characteristics of gonadotrophin secretion on day 22, nor of ovarian activity on day 26, were significantly influenced by the level of feeding during pregnancy or lactation.     

Neonatal hyperleptinaemia programmes anxiety-like and novelty seeking behaviours but not memory/learning in adult rats

Leptin treatment during lactation programmes for leptin resistance at adulthood, evidenced by hyperleptinaemia, hyperphagia and overweight. Since leptin is known to affect stress response, emotional behaviour and memory/learning performance, the objective of the present study was to evaluate whether neonatal hyperleptinaemia programmes anxiety-like and novelty-seeking behaviours as well as memory/learning in adult male rats. During the first 10 days of lactation (from PN1 to PN10), pups were s.c. injected once per day with either 50 μL of saline (SAL) or murine leptin (LEP — 8 μg/100 g of body mass, saline diluted). Serum leptin was assessed at PN10 and at PN150. Two separate experiments were carried out: 1) experiment one: at PN137, 29 SAL and 30 LEP rats were tested in the elevated plus-maze (EPM) and, at PN142, their behaviour was assessed in the hole board (HB) arena; 2) experiment two: at PN140, a different group of rats consisting of 53 SAL and 56 LEP animals were tested in the radial arm water maze (RAWM). Serum leptin concentration was higher in the LEP group at PN10 and at PN150. LEP animals spent significantly less time in the open arms of the EPM. Furthermore, the number of nose-pokes in the HB arena was higher in LEP rats. There were no differences between groups regarding latency to find the hidden platform in the RAWM. Our results suggests that a central mechanism of leptin resistance at adulthood, caused by neonatal hyperleptinaemia, is associated with an increased level of anxiety and also that it intensifies novelty seeking-behaviour.     

Decreased plasma leptin levels in lean and obese Zucker rats after treatment with the serotonin reuptake inhibitor fluoxetine.

Leptin inhibits feeding, stimulates thermogenesis and decreases body weight. Serotonin reduces food intake when injected into the hypothalamus and may interact with other neurotransmitters in the control of appetite. We therefore investigated the effects of the serotonergic drug fluoxetine, which inhibits serotonin reuptake, on food intake and plasma leptin levels in lean and obese Zucker rats. Fluoxetine significantly reduced food intake in lean and obese rats, both acutely after a single injection (10 mg/kg) and during continuous subcutaneous infusion (10 mg/kg/day for 7 days). Plasma leptin levels were reduced after both 4 hours and 7 days of fluoxetine administration in lean and after 7 days in fatty rats (all p<0.01). We have previously suggested that serotonin may decrease food intake by inhibiting neuropeptide Y neurones, and we further suggest that it also inhibits leptin, possibly by an effect on white adipose tissue.     

Programmed obesity in intrauterine growth-restricted newborns: modulation by newborn nutrition

The degree of nutrient enhancement during the newborn period may modulate programming of appetite-regulating hormones, body composition, and propensity to adult obesity in intrauterine growth-restricted (IUGR) newborns. Pregnant rats received, from day 10 to term gestation and throughout lactation, ad libitum food (AdLib) or 50% food restriction (FR) to produce IUGR newborns. AdLib vs. FR offspring were studied at day 1, and, to create two distinct groups of newborn catch-up growth (immediate, delayed) among the IUGR newborns, cross-fostering techniques were employed. The four groups of pups at 3 wk were IUGR immediate catch-up growth (FR/AdLib), IUGR delayed catch-up growth (FR/FR), control (AdLib/AdLib), and lactation FR control (AdLib/FR). From 3 wk to 9 mo, all offspring had AdLib rat chow. Maternal FR during pregnancy resulted in IUGR pups (6.0 +/- 0.3 vs. 7.1 +/- 0.3 g, P < 0.01) with decreased leptin (0.66 +/- 0.03 vs. 1.63 +/- 0.12 ng/ml, P < 0.001) and increased ghrelin (0.43 +/- 0.03 vs. 0.26 +/- 0.02 ng/ml, P < 0.001). Maternal FR during lactation (FR/FR) further impaired IUGR offspring growth at 3 wk. However, by 9 mo, these pups attained normal body weight, percent body fat, and plasma leptin levels. Conversely, IUGR offspring nursed by AdLib dams (FR/AdLib) exhibited rapid catch-up growth at 3 wk and continued accelerated growth, resulting in increased weight, percent body fat, and plasma leptin levels. Thus the degree of newborn nutrient enhancement and timing of IUGR newborn catch-up growth may determine the programming of orexigenic hormones and offspring obesity.     

Hypothalamic hypocretin/orexin and neuropeptide Y: divergent interaction with energy depletion and leptin

The aim of this study was to measure the effects of chronic leptin treatment on two orexigenic peptides present in the hypothalamus namely hypocretin/orexin and neuropeptide Y (NPY). For this purpose, recombinant murine leptin (0.2 mg/rat/day) or saline were injected intraperitoneally in Long-Evans rats for 7 consecutive days. Food intake (-8%; p < 0.002) and body weight gain (23.7 +/- 1 vs 31.5 +/- 1.3 g; p < 0.003) were significantly lower in leptin-treated rats than the saline-treated rats. NPY concentrations did not change significantly in any of the microdissected brain areas including the arcuate and paraventricular nuclei. Orexin A concentration in the lateral hypothalamus was significantly decreased by the leptin treatment (-68%; p < 0.01). A smaller decrease (-46%; p < 0.04) was also noted in saline-treated rats pairfed to the level of the leptin-treated rats. We conclude that orexin/hypocretin could be considered as a new relay for leptin in the central nervous system. Its variation in case of lower energy supply observed in pairfed rats could constitute an alerting system for the brain and therefore considered as the first step in the establishment of defense mechanisms against energy depletion.     

Circulating leptin levels in newborn rats: a significant post- natal developmental effect,independent of dietary polyunsaturated fat levels

Leptin expression exhibits developmental and dietary regulation, but it is unknown whether there is an interaction of the regulation by dietary fat and postnatal development. The purpose of this study was to test the effect of different levels of dietary polyunsaturated fat on circulating leptin levels at different post-natal developmental stages. Pregnant (Sprague-Dawley) rats consumed from day 15 of pregnancy through day 9 of lactation a low fat, (11% of energy; LF) polyunsaturated safflower oil diet. From day 9 of lactation, dams and their respective pups were fed low, moderate (40% of energy; MF) or high (67% of energy; HF) polyunsaturated safflower oil diets to full maturation (56 days). Diets were iso-energetic and iso-nitrogenous. Milk fatty acid content reflected the mothers and pups diet, with 15 to 100 fold less C10:0 and 2.6 to 3.3 fold more C18:2 in MF and HF groups compared to LF diet. In newborn rats through post-natal day 56, levels of polyunsaturated fat in mothers' milk and mothers/pups diet had no effect on the levels of circulating leptin. The post-natal development period significantly affected circulating leptin levels (p < 0.001, 15 days = 56 days > 21 days > 28 days). In summary, the developmental postnatal stage regulates leptin levels, independently of the polyunsaturated fat levels in the diet.     

AAV mediated expression of anti-sense neuropeptide Y cRNA in the arcuate nucleus of rats results in decreased weight gain and food intake

Neuropeptide Y (NPY) is the most potent stimulant of feeding when administered by intracerebroventricular injection. Despite this, there is conflicting evidence as to its importance in the regulation of daily food intake and energy balance. It has been suggested that whilst it is important in the response to starvation it has little role in the regulation of daily food intake. To investigate the role of NPY in the regulation of food intake, anti-sense cRNA to NPY was expressed in the arcuate nucleus of adult male rats. The anti-sense NPY (AS-NPY) construct was initially tested in vitro and there was a decrease of approximately 50% in NPY release from anti-sense treated cells compared to controls (16.3 +/- 2.0 fmol/L [AS-NPY] vs 37.3 +/- 7.7 fmol/L [control], mean +/- SEM p < 0.05). NPY release from hypothalamic explants from anti-sense injected animals was decreased by over 50% compared to those from controls at both 15 and 20 days after AAV injection (15 days 42% +/- 6.5% [AS-NPY] vs 100% +/- 36% [control], 20 days 41% +/- 6% [AS-NPY] vs 100% +/- 27% [control] mean+/-SEM, p < 0.05). In a study lasting for 50 days, weight gain was significantly lower in anti-sense injected animals from day 16 (day 16: 6.25 +/- 1.10 g [AS-NPY] vs 9.42 +/- 0.65 g [control] mean +/- SEM, p < 0.05) and remained so until the end of the study when they had gained approximately 40% less weight than controls (day 50: 52.0 +/- 9.6 g [AS-NPY] vs 82.0 +/- 6.3 g [control] mean +/- SEM, p < 0.01). Cumulative food intake was significantly lower in the anti-sense injected animals from day 23 (day 23: 225.8 +/- 1.9 g [AS-NPY] vs 250.6 +/- 8.7 g [control], mean +/- SEM, p < 0.05) and remained so until the end of the study (day 50: 834.5 +/- 14.8 g [AS-NPY] vs 926.0 +/- 31.7 g [control], mean +/- SEM, p < 0.05). Similarly mean daily food intake was also reduced in the anti-sense injected animals (days 7-14: 24.9 +/- 0.4 g/day [AS-NPY] vs 27.2 +/- 0.4 g/day [control], mean +/- SEM, p < 0.01). These data are supportive of a role for NPY in the regulation of daily food intake as well as in response to starvation.