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APPETITE, OBESITY, DIGESTION, AND METABOLISM
1Department of Neurology and Neurosciences, New Jersey Medical School, University of Medicine and Dentistry, Newark, New Jersey; 2Department of Pharmacology, Merck Research Laboratories, Rahway, New Jersey; and 3Neurology Service, Department of Veterans Affairs New Jersey Health Care System, East Orange, New Jersey
Submitted 25 October 2006 ; accepted in final form 5 January 2007
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ABSTRACT |
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diet-induced obesity; hypothalamus; development; plasticity; melanocortin
Clearly, many factors might have contributed to altered neural development during this prolonged study period. For this reason, we undertook the present studies in which rats from obese and lean DIO and DR dams were assessed morphometrically at both postnatal day (PND) 16 and 6 wk of age. We also assessed the expression of a number of hypothalamic neuropeptides and neuropeptide receptors involved in the regulation of energy homeostasis at 6 wk of age with the prediction that maternal obesity would further reduce the already attenuated leptin and insulin signaling of the chow-fed, selectively bred DIO rats (12, 17, 18). Unexpectedly, we found that 6-wk-old offspring of obese DIO dams had profiles of leptin (Lepr-b), insulin (InsR) and melanocortin-4 receptor (MC4R) mRNA expression, which suggested exactly the opposite was true. For this reason, we undertook a second series of studies to test the functional competence of leptin and melanocortin signaling by assessing the anorectic and brown adipose tissue (BAT) thermogenic responses to intracerebroventricular injections of leptin and the melanocortin agonist, Melanotan II (MTII), in offspring of lean DR and lean and obese DIO dams.
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METHODS |
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Animal usage was in compliance with the Institutional Animal Care and Use Committee of the East Orange Veterans Affairs Medical Center. All studies were carried out in keeping with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society (1). Dams used were from our two colonies of rats selectively bred for their propensity to develop either DIO or DR (16). These colonies were originally derived from outbred Sprague-Dawley rats (Charles River Laboratories). Rats were kept at 23–24°C on a reversed 12:12-h light-dark cycle with lights off at 1000 with food and water available ad libitum. Two sets of studies were carried out.
Study 1
Dams and breeding. This study used 4 sets of dams: 1) lean DR dams (n = 11) were fed Purina rat chow (#5001) containing 3.75 kcal/g with 23.4% of total calories as protein, 4.5% as fat, and 72.1% as carbohydrate, which is primarily in the form of complex polysaccharide; 2) obese DR dams (n = 8) were made obese with chocolate Ensure (Ross Products), which is a liquid diet containing 1.06 kcal/ml with 14% of the metabolizable energy content as protein, 22% as fat, and 64% as carbohydrate. We previously showed that DR dams become obese and hyperinsulinemic as adults on this diet (20); 3) Lean DIO dams (n = 7) were fed chow; 4) obese DIO dams (n = 8) were made obese on a HE diet that contains 4.41 kcal/g with 17% of total calories as protein, 32% as fat, and 51% as carbohydrate, 50% of which is sucrose (13) (Research Diets #D12266B). After 1 mo on their respective diets, all dams underwent tail bleeding for plasma glucose, insulin, and leptin levels and then were mated with males of the same genotype. All litters were adjusted to 10 pups (5 male: 5 female). Dams were kept on their respective diets throughout gestation and weaning. Additional blood samples were drawn during the second week of gestation and second week of lactation.
Offspring manipulations. Because hypothalamic pathways mediating energy homeostasis continue their development well into the third postnatal week in the rodent (2, 8, 29), we chose to examine offspring at PND 16. On PND 16, two male offspring from each litter were weighed and killed by decapitation between 1000 and 1300. Trunk blood was collected for insulin and leptin analysis. Fat pads (retroperitoneal, perirenal, mesenteric, epididymal, and inguinal) were dissected and weighed. Remaining male pups were weaned to chow five days later. We studied a second cohort of rats at 6 wk of age because rats of this age are in a stage of rapid growth which occurs just prior to puberty, an age at which DIO rats already have reduced central leptin signaling (17). After an overnight fast, 6-wk-old offspring underwent an oral glucose tolerance test. Two days later, chow was removed at 0800, and rats were killed by decapitation between 1000 and 1300. Trunk blood was collected for insulin and leptin analysis. Brains were quickly removed, frozen on powdered dry ice, and stored at –80°C for later assay by quantitative real-time RT-PCR (QPCR). Fat pads (retroperitoneal, perirenal, mesenteric, epididymal, and inguinal) were dissected and weighed.
Oral glucose tolerance test. Following an overnight fast, a baseline blood sample (0.25 ml) was collected from tail blood into heparinized tubes for glucose and insulin assays. Rats were then gavaged with 0.5 g/kg glucose, and blood for insulin and glucose was sampled via tail nick at 15, 30, 60, 90, and 120 min.
Assays of insulin, leptin, and glucose. Plasma insulin and leptin levels were analyzed with radioimmunoassay kits (Linco), using antibodies to authenticate rat insulin and leptin, respectively. Whole blood was used to measure glucose using a glucometer (Accuchek).
Hypothalamic neuropeptide and receptor mRNA assays by QPCR. Frozen brains were cut on a cryostat at –12°C into two 300-µm sections centered on the hypothalamic paraventricular nucleus (PVN) and at the midpoint of the arcuate nucleus (ARC). The latter section also contains the midpoint of the ventromedial nucleus (VMN) and dorsomedial nucleus (DMN) through its compact portion and the lateral hypothalamus (LH). Cut sections were placed in RNA Later (Ambion) until micropunched. Micropunches of the PVN, ARC, VMN, DMN, and LH were performed by modifications (17, 22) of the method of Palkovits (28). Briefly, brain slices were placed on the base of a stereotaxic frame and visualized under an operating microscope. The syringe to which the punches were attached was mounted on the stereotaxic arm, and the punches were made under microscopic guidance. Micropunched brain nuclei were sonicated in a guanidinium thiocyanate solution and purified using magnetic beads (Ambion MagMax-96). Quantitation of mRNA was carried out by QPCR as previously described (17). Briefly, genomic DNA was removed with DNase, mRNA was reverse-transcribed with random hexamer priming using Superscript-3 (Invitrogen) and treated with RNaseH (Ambion). Primer sets for each mRNA were designed by reference to published sequences and their specificity was verified using GenBank. Primers and their sequence-specific 6-Carboxyfluorescein-labeled probes prepared by Applied Biosystems (Table 1) were sequenced and then quantified with an Applied Biosystems 7700 real-time PCR system set for 40 PCR cycles. Standard curves were generated from serially diluted pooled samples for each probe and for constitutively expressed mRNA (cyclophilin) to control for differences in amplification efficiency. Results were calculated from the standard curves relative to cyclophilin mRNA levels in the same samples.
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Dams and breeding. Because expression of hypothalamic neuropeptide and receptor signaling mRNA's were comparable between offspring of lean, chow-fed, and obese, Ensure-fed DR dams in study 1, we used only three sets of dams in this second set of studies to reduce the number of offspring tested: 1) lean DR dams were fed chow (n = 6); 2) lean DIO dams were fed chow (n = 7); 3) obese DIO dams were fed HE diet beginning 1 mo before impregnation (n = 6).
Leptin effects on food intake and BAT temperature in offspring.
At 6 wk of age, male offspring of lean DR and lean and obese DR dams were anesthetized with chloropent (0.3 mg/100 g ip) and were stereotaxically implanted with indwelling unilateral 26-gauge cannulas aimed at the right lateral ventricle (AP –1.2 mm bregma; lateral 1.4 mm, dura 3.4 mm). Rats were allowed 5 days for recovery, during which time they were handled daily and given sham injections. On day 5, angiotensin (1 µg in 0.2 µl) was administered and those rats that consumed 
5 ml were then surgically implanted with PDT-4000 E-Mitter transponders (MiniMitter) under light isoflurane anesthesia. Transponders were placed below the interscapular BAT pad and sutured in place. This telemetry system continuously monitors both temperature (°C) and activity (arbitrary units) data, which are acquired and stored using Vital View, an integrated software and hardware system specifically designed for controlling data acquisition. Rats were kept at 23–24°C on a reversed 12:12-h light-dark cycle with lights off at 0900. On the days of testing, food was removed at 0700 and DR (n = 5) and lean (n = 5) and obese DIO (n = 6) offspring were injected intracerebroventricularly between 0830 and 0930 with either 5 µl saline or leptin (1 and 10 µg in 5 µl). Food was returned, and food intake and BAT temperature responses were measured simultaneously over the 4 and 24-h periods following each treatment. Injections with saline and both leptin doses were carried out sequentially in all rats with 3-day rest periods between each dose.
MTII effects on food intake and BAT temperature. One week following leptin administration, using the same protocol as that used for leptin, all rats received either 5 µl vehicle or 0.25 and 0.5 nmol MTII, a nonselective melanocortin agonist (4), in 5 µl (Peninsula Laboratories). All rats received saline and both doses of MTII with 5 days rest between treatments.
Statistics.
Total food intake; area under the curve (AUC) for glucose and insulin during the oral glucose tolerance test and BAT temperature changes; insulin sensitivity index; terminal body and fat depot weights; and plasma glucose, insulin, and leptin levels were first compared by two-way ANOVA (maternal genotype x phenotype). Additional comparisons were made by one-way ANOVA by experimental group. When significant intergroup differences were found by ANOVA (P 
0.05), post hoc comparisons were carried out by Bonferroni analysis. All statistical analyses, including rejection of statistical outliers, were determined using Systat statistical software. Feed efficiency was estimated by dividing the weight gain by the number of calories ingested over the same period of observation. Food intake effects following leptin and MTII administration were compared by paired t-test for each rat's food intake vs. its own vehicle-treated food intake. BAT temperature responses over 4-h and 24-h periods following intracerebroventricular vehicle, leptin, and MTII injections were determined by calculating the change from baseline (average BAT temperature between 0800 and 0830) as AUC using the trapezoidal rule. The insulin sensitivity index was calculated from the formula of Matsuda and Defronzo (23) [10,000/square root of (fasting glucose x fasting insulin) x (mean glucose x mean insulin during an oral glucose tolerance test)] (23).
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RESULTS |
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Dam body weight gain and plasma hormone levels. Similar to previous studies of these selectively bred rats (16), DIO dams were heavier compared with DR dams before they were begun on the HE diet. After 1 mo on their respective diets, DR dams fed chow gained the same amount of body weight as the chow-fed DIO dams, while the DR dams fed Ensure gained the same amount of weight as DIO dams fed HE diet (Table 2). Thus, we use the terms "lean and obese DR and DIO" to describe the respective dams and their offspring. The body weight gain of both obese DR and DIO dams was threefold greater than their respective chow-fed lean controls. The obese DIO dams gained 237% more weight and had 155% higher plasma insulin levels than lean DIO dams. Obese DR dams had comparable plasma insulin levels to obese DIO dams, and these were, in turn, 200% greater than those in lean DR and DIO dams. Leptin levels were the highest in the obese DIO and DR dams, intermediate in the lean DIO, and lowest in the lean DR dams. Glucose levels in the obese DR dams were comparable to those in both obese DIO and lean DR dams and 15% higher than those in lean DIO dams. At 2 wk of gestation, obese DIO dam body weights were 11, 14, and 23% higher than those in lean DIO, obese DR, and lean DR dams, respectively. In addition, there was a significant interaction of genotype and maternal phenotype on body weight gain [F (1, 29) = 8.038; P = 0.008]; obese DR dams gained the least amount of weight compared with all of the other groups. Plasma insulin levels did not differ among the groups while both glucose [F (1, 30) = 8.544; P = 0.007] and plasma leptin [F (1, 28) = 14.763; P = 0.001] levels showed a genotype x maternal phenotype interaction. Leptin levels were highest of all groups in obese DIO dams, were comparable between obese DR and lean DIO, and were lowest in lean DIO dams. At delivery, all dams were maintained on their respective diets. During the first 2 wk after delivery, obese DIO dams lost body weight, so that their weights became comparable to those of obese DR and lean DIO dams. On the other hand, obese DR dams continued to gain weight during lactation and remained heavier than the lean DR dams at 2 wk postgestation. Commensurate with their loss of body weight, leptin levels fell 73% in obese DIO dams during 2 wk of lactation, although their levels still remained higher than all of the other groups. Insulin and glucose levels did not differ significantly among the groups at this time.
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Leptin effects on food intake and BAT temperature in 6-wk-old chow-fed offspring of lean DR and lean and obese DIO dams. When rats were injected intracerebroventricularly with vehicle at dark onset when they would normally begin to feed, offspring of lean DR dams ate more over the ensuring 4 h than did offspring of either obese or lean DIO dams (Fig. 2A). Although both the 1-µg and 10-µg doses of leptin reduced food intake in offspring of lean DR dams compared with their vehicle-injected levels, neither dose affected intake in offspring of lean or obese DIO dams. This result was in keeping with our previous studies demonstrating a reduced anorectic response to leptin in preobese DIO rats (15, 17). Over the full 24-h period following intracerebroventricular vehicle injections, there were no differences in food intake among the groups. However, not only did both the 1-µg and 10-µg doses of leptin reduce intake in offspring of lean DR dams relative to their vehicle control levels, it also reduced intake in offspring of obese DIO dams, although to a lesser degree than in the former group (Fig. 2B). In no case did leptin alter intake in offspring of lean DIO dams.
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When 1-µg and 10-µg doses of leptin were injected at dark onset, there were significant intergroup differences found by repeated-measures ANOVA for the BAT temperature responses over the first 4 h [Fig. 2D; F (4,20) = 5.651; P = 0.003]. BAT temperatures rose rapidly over the first 20 min and remained elevated for most of the first 4 h after the 1-µg leptin injections in offspring of DR and obese DIO dams (Fig. 2C). However, the onset of the leptin-induced increase in BAT temperature was considerably delayed and not as sustained in offspring of lean DIO dams over this period (Fig. 2C). Although there were no significant differences in total AUC among the groups over 4 h following these 1-µg injections, only the offspring of obese DIO dams had a significant increase in their 4-h BAT temperature compared with their own vehicle-injected control AUC values (Fig. 2D). In addition, only the offspring of lean DR dams showed a significant increase in 4-h BAT temperature following 10 µg intracerebroventricular leptin. On the other hand, offspring of obese DIO dams had no increase above their own vehicle controls at the 10-µg dose of leptin. The rise in BAT temperature at 4 h after 1 µg leptin in the offspring of obese DIO dams could not be attributed to altered energy intake as that dose had no effect on intake in those rats (Fig. 2A). Although offspring of obese DIO dams had higher BAT temperature AUCs over the 24 h following vehicle injections than offspring of lean DR dams, leptin had no overall effect on 24-h BAT temperature in any group (Fig. 2E).
MTII effects on food intake and BAT temperature in 6-wk-old chow-fed offspring of lean DR and lean and obese DIO dams. As with the injection of vehicle for the leptin studies, offspring of lean DR dams ate more than the lean DIO and obese dams over the 4 h after vehicle injection in the MTII studies (Fig. 3A). However, as opposed to the differential effects of leptin on food intake among the groups, all 3 groups responded by decreasing their food intake by comparable amounts at both 4 h and 24 h after both 0.25 and 0.5 nmol MTII relative to their own vehicle-control levels (Fig. 3, A and B). Following 0.25 nmol intracerebroventricular MTII, there was a rapid rise in BAT temperature in all groups over the first 5–10 min following injection (Fig. 3C). This increase was largely sustained over the first 4 h in offspring of DR and obese DIO dams but had a transient postinjection dip from 90 to 200 min in offspring of lean DIO dams (Fig. 3C). Despite this difference in the patterns of response, the 4-h BAT temperature AUC values did not differ significantly among groups at this dose of MTII (Fig. 3D). Although there was a tendency for BAT temperature to increase above vehicle-injected control levels in all groups injected with 0.25 nmol MTII over 4 h, this did not reach statistical significance over 4 h (Fig. 3, D and E). Neither were there significant BAT thermic effects of either dose of MTII over 24 h. Thus, MTII had a comparable anorectic response in all groups but little effect on BAT temperature. It is certainly possible that there would have been a more robust temperature response in all groups had MTII not simultaneously suppressed their food intake.
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DISCUSSION |
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Reports that leptin-deficient mice have reduced ARC proopiomelanocortin mRNA, which is normalized by leptin treatment (24, 31) and that the anorectic effects of leptin are blocked by a melanocortin receptor antagonist (32), suggest that the melanocortin system is an important downstream catabolic effector of leptin's actions. Here, we found that offspring of lean DIO dams did not differ from lean DR offspring in their expression of MC3R or MC4R mRNA in the ARC, VMN, or PVN. This is in keeping with our previous studies showing that there were also no differences in binding to hypothalamic MC3/4R between lean chow-fed DR and DIO rats at 13 wk of age (12). On the other hand, despite the fact that VMN expression of MC4R mRNA was several-fold higher in offspring of obese DIO dams, they were not more responsive to the anorectic or thermogenic effects of MTII than other offspring. This suggests that VMN MC4R alone is not a major determinant of the catabolic effects of melanocortins. However, both hypothalamic melanocortin (27) and insulin signaling (3, 26) are important in the regulation of peripheral insulin sensitivity. Also, selective reductions in both VMN InsR and melanocortin receptor mRNA expression are associated with reduced peripheral insulin sensitivity in offspring of DR dams cross-fostered to obese DIO dams (7). Thus, it is possible that the elevated VMN MC4R and InsR mRNA expression made an important contribution to the increased insulin sensitivity of 6-wk-old offspring of obese DIO vs. lean DIO dams.
In evaluating the thermogenic effects of intracerebroventricular leptin and MTII, we used BAT temperature as a surrogate. Because of the large number of intracerebroventricular injections required to assess this function at multiple doses in the same animals, we measured both food intake and BAT temperature at the same time. We found that vehicle-injected offspring of obese DIO dams had consistent 24-h (Figs. 2E and 3E) and less consistent 4-h (Fig. 2D vs. 3D) increases in BAT temperature compared with offspring of either lean DIO or DR dams. This raised, "basal" BAT thermogenesis occurred even though offspring of obese DIO dams ate less than DR offspring over 4 h, while all groups had comparable energy intake over 24 h (Figs. 2B and 3B). This suggests that the offspring of obese DIO dams had increased diet-induced thermogenesis compared with the other groups under basal conditions.
Leptin has been well documented to increase sympathetic activity (11) and thermogenic capacity of BAT (30). Following intracerebroventricular injection, 1 µg of leptin increased BAT temperature only in the offspring of obese DIO rats (Fig. 2D), despite the fact that this dose had no effect on their energy intake (Fig. 2A). It is possible that the DR offspring might have had a greater increase in their BAT temperature responses had leptin not simultaneously suppressed their intake. Thus, we can draw no firm conclusions about the relative thermic effects of leptin in BAT between offspring of DR and obese DIO dams. However, the important point is that offspring of obese DIO dams still had a greater 4-h increase in BAT temperature than did lean DIO dam offspring following the 1-µg leptin injections. This occurred despite the fact food intake was suppressed in neither group at this dose over this same period. Taken together, with the increased 24-h anorectic effect of leptin at both leptin doses, these data firmly support the contention that 6-wk-old offspring of obese DIO dams have a greater diet-induced thermogenesis under basal conditions compared with both of the other groups and that they were more sensitive to the thermogenic and anorectic effects of leptin than the offspring of lean DIO dams. Given the importance of ARC Lepr-b in energy homeostasis (25), it is likely that the increased ARC Lepr-b expression of obese DIO dam offspring was an important contributing factor to their enhanced leptin-induced anorexia and BAT thermogenesis. Unfortunately, the mechanism underlying this increase in sensitivity at 6 wk of age is unclear at present and must await further studies. Similarly, we have no ready explanation for the fact that the increased thermic effect of leptin was seen after 1 µg but not 10 µg of leptin in offspring of obese DIO dams.
As opposed to leptin, there were no statistically significant BAT temperature responses following either 0.25 nmol or 0.50 nmol (icv) MTII, although both doses reduced energy intake to a comparable degree in all groups. This lack of a BAT thermogenic response was surprising, given the fact that intracerebroventricular MTII increases sympathetic nerve activity, even in anesthetized rats (10). Although MTII did appear to increase BAT temperature in all groups over the first 4 h after intracerebroventricular injection (Fig. 3C), this rise did not quite reach statistical significance compared with each animals' own vehicle-injected control levels (Fig. 3D). It is possible that the nonsignificant increase in BAT temperature that was seen in all groups over the first 4 h following MTII injections might have reached significance had the animals been assessed in a fasting condition. Nevertheless, because MTII had the same anorectic effect in all groups, it is likely that there were no significant intergroup differences in the BAT thermic effects after stimulation of central melanocortin receptors.
One important caveat of the current studies is that the use of different diets to make DR (Ensure) and DIO dams (HE diet) obese might have contributed to some of differences between offspring of obese and lean DIO dams. Pups begin to eat the maternal diet toward the end of lactation, so that exposure of pups or dams to differing diets and/or a contrast effect between early intake of the maternal diet and the postweaning consumption of chow might have affected some of the outcome variables. Although admittedly not ideal, we used diets of differing fat, carbohydrate, and caloric density to produce obesity in DR and DIO dams as an expedient to reduce the number of animals used. This decision was based on our previous demonstration that diet was a major determinant of neither obesity of offspring nor of altered development of hypothalamic monoamine pathways involved in the regulation of energy homeostasis (14, 19). In those studies, both DIO and DR dams were fed a HE diet through gestation and lactation. Although offspring of obese DIO dams fed a HE diet became more obese than all other groups, offspring of lean DR dams fed HE diet did not differ from offspring of lean DR or DIO dams fed chow in any way. Thus, it is unlikely that differences among groups seen here were due to the lack of a control group of offspring whose dams were fed a HE diet.
In those same studies (14, 19), offspring of DR dams made obese by feeding them Ensure did not become obese as adults, but they did exhibit some abnormalities of hypothalamic monoamine pathway development. For this reason, we used Ensure to make DR dams obese with the expectation that, while their offspring would not become more obese, the exposure to Ensure and/or maternal obesity might alter their expression of hypothalamic neuropeptide and receptor mRNAs. In fact, offspring of obese DR dams fed Ensure exhibited no differences in any of these measures compared with offspring of lean DR dams fed chow. Thus, on the basis of our previous studies (14, 19) and the present results, which demonstrated no apparent effect of diet alone, we believe that the interactions of maternal genotype and obesity were the major determinants of the outcome differences seen among the groups at 6 wk of age.
In conclusion, there is a critical interaction between genetic background and maternal obesity that affects the development of hypothalamic receptor, neurotransmitter, and neuropeptide systems involved in the regulation of energy homeostasis. Although offspring of obese DIO dams become more obese than offspring of lean DIO and obese and lean DR dams as adults (Fig. 4) (19), we found here that they had comparable adiposity and body weight but were more sensitive to the central effects of leptin and peripheral effects of insulin when fed chow from weaning to 6 wk of age. Even though such increased sensitivity should predispose them to become less, rather than more, obese as adults, this does not occur. Hence, there is a potential window of opportunity where an environmental intervention might prevent these highly obesity-prone rats from realizing their full potential. The current results suggest that future studies should focus on this transitional period when juvenile offspring of obese DIO dams appear to be relatively obesity resistant and at the onset of adulthood when the full expression of their obesity occurs. It is possible that elucidation of the factors that cause this transition will allow us to prevent the development of adult-onset obesity in individuals who are both genetically and developmentally predisposed to such a fate.
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ACKNOWLEDGMENTS |
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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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This article has been cited by other articles:
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  C. M. Patterson, A. A. Dunn-Meynell, and B. E. Levin Three weeks of early-onset exercise prolongs obesity resistance in DIO rats after exercise cessation Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R290 - R301. [Abstract] [Full Text] [PDF]
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 B. E. Levin Why some of us get fat and what we can do about it J. Physiol., September 1, 2007; 583(2): 425 - 430. [Abstract] [Full Text] [PDF]
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