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Obesity is a chronic metabolic disorder affecting half a billion people worldwide. Major difficulties in managing obesity are the cessation of continued weight loss in patients after an initial period of responsiveness and rebound to pretreatment weight. It is conceivable that chronic weight gain unrelated to physiological needs induces an allostatic regulatory state that defends a supranormal adipose mass despite its maladaptive consequences. To challenge this hypothesis, we generated a reversible genetic mouse model of early-onset hyperphagia and severe obesity by selectively blocking the expression of the proopiomelanocortin gene (Pomc) in hypothalamic neurons. Eutopic reactivation of central POMC transmission at different stages of overweight progression normalized or greatly reduced food intake in these obesity-programmed mice. Hypothalamic Pomc rescue also attenuated comorbidities such as hyperglycemia, hyperinsulinemia, and hepatic steatosis and normalized locomotor activity. However, effectiveness of treatment to normalize body weight and adiposity declined progressively as the level of obesity at the time of Pomc induction increased. Thus, our study using a novel reversible monogenic obesity model reveals the critical importance of early intervention for the prevention of subsequent allostatic overload that auto-perpetuates obesity.
Obesity is a chronic metabolic disorder that affects more than 500 million adults and 43 million children under the age of 5 worldwide (1). Overweight predisposes individuals to numerous comorbidities, including type 2 diabetes mellitus, hypertension, cardiovascular disease, and certain forms of cancer (2). The causes of obesity are complex, because environmental, social, and genetic factors converge to produce a condition in which energy intake consistently exceeds energy expenditure, leading to the storage of excess calories as fat.
Unlike homeostatic processes that operate around narrowly defined set points defended solely by the autonomic nervous and endocrine systems, body weight regulation and fat storage are allostatic metabolic processes by which voluntary actions, including foraging and food intake, are engaged in anticipation of future caloric deficits including foraging and food intake. Because body growth is an enduring process across the first stages of a mammal’s life, the allostatic state is evident from early development of the fetus and continues postnatally until completion of sexual maturation. Thereafter, body weight is typically defended around a relatively more stable set point (3), with the exception of intervening physiological conditions such as pregnancy, lactation, or hibernation. These latter states reengage the allostatic energy balance system to trigger short-term feed-forward mechanisms that promote an increase in food intake, fat accumulation, and body weight. All of these parameters revert to previous values once less-demanding environmental conditions allow reduced caloric intake. Similarly, voluntary overfeeding can lead to a great increase in body weight that gradually returns to preexisting levels when normal feeding is resumed (4, 5). However, this is not generally true in obese patients, who demonstrate an inability to completely achieve a normal weight and exhibit high recidivism after dieting, in large part due to compensatory and persistent reductions in metabolic rate (6–8). These problems worsen in extreme obesity (BMI >40 kg/m2), a condition that is increasing alarmingly (9, 10). Diet-induced obesity in rats and mice can also permanently elevate body weight set point after the animals are switched back to normal chow (11–13). However, unlike the allostatic changes in metabolism that occur during pregnancy or hibernation to promote “viability through change” (14), chronic overfeeding unrelated to physiological needs generates an allostatic state that slowly leads to obesity and is ultimately maladaptive to the health of an organism.
The brain is a main regulator of energy balance and receives peripheral signals of energy status in the form of circulating nutrients and hormones, such as leptin, combined with afferent neural signals relayed through the vagus nerve (15). Two populations of neurons within the arcuate nucleus (Arc) of the hypothalamus sense and respond to most of these signals (16). Orexigenic agouti-related peptide (AgRP) and neuropeptide Y (NPY) neurons are inhibited by leptin, while proopiomelanocortin (POMC) and cocaine- and amphetamine-related transcript (CART) neurons are stimulated by leptin. Pomc is highly expressed in the pituitary gland and hypothalamus of all vertebrates (17) by means of two independent cis-acting enhancer modules; a proximal promoter that controls pituitary Pomc expression and a distal module that is necessary for hypothalamic Pomc expression (18). There is also a small number of neurons in the nucleus tractus solitarius (NTS) of the brainstem that express much lower levels of Pomc mRNA and whose physiological function is poorly understood. Among hypothalamic POMC products are the anorectic melanocortin peptides α- and β-MSH and the opioid peptide β-endorphin, which are released in response to metabolic peripheral signals (19, 20). Genetic inactivation of POMC in humans and rodents results in severe hyperphagia, early-onset obesity, and profound adrenal insufficiency (21, 22), whereas mutations in the gene encoding central melanocortin receptor 4 (MC4R) are the most common monogenic disorders causing obesity (16).
Because of the high prevalence of overweight and the severity of its comorbidities (1, 2), it is imperative to understand the factors associated with treatment refractoriness. Our principal hypothesis is that energy homeostasis can be disturbed acutely within a certain range without major consequences for the organism. However, when chronic perturbations occur, such as severe hyperphagia or sustained low energy expenditure, the elastic limits of the regulatory system are exceeded and normal weight can no longer be achieved due to persistent adaptations. To test this hypothesis, we generated a reversible knockout mouse model of early-onset extreme obesity by inhibiting Pomc expression in the hypothalamus through a genetic switch that allows subsequent reactivation of gene expression. These mice develop obesity while consuming a standard low-fat chow, and therefore the genetic model avoids the conundrum of permanent changes in dopaminergic reward centers induced by high-fat or high-sucrose diets, even after return to food with low hedonic value (23), as typically occurs in diet-induced obesity models. We rescued hypothalamic Pomc in mice with different degrees of overweight and found marked improvements in food intake, body weight, fat deposits, and fasting glucose even in cases of extreme obesity. However, increasing levels of overweight at the time of genetic rescue progressively reduced the ability of mice to achieve a normal body weight.
ArcPomc– mice carry a mutant Pomc allele harboring an upstream loxP-flanked neo cassette that selectively prevents neuronal transcription in the hypothalamic Arc but not in pituitary cells (Figure (Figure1,1, A and B). The lack of hypothalamic Pomc expression in arcPomc–/– mice led to early-onset extreme obesity, readily detected by age 5 weeks (P < 0.001 versus Pomc+/+ and arcPomc+/– littermates, 1-way ANOVA [OWA], Figure Figure1C).1C). However, compound arcPomc–/– mice carrying a Pomc-EGFP transgene (19) expressed the EGFP reporter molecule in the same number, and with a similar morphology and spatial location, of neurons within the Arc as Pomc+/+ mice (Figure (Figure1B).1B). The EGFP-positive neurons also projected widely throughout the brain to areas normally innervated by POMC neurons (data not shown). These results indicate that a subpopulation of Arc neurons is differentiated with the transcriptional machinery capable of normally activating Pomc and projects to relevant target areas despite the absence of POMC-derived peptides throughout prenatal development and into postnatal life. ArcPomc–/– mice were hyperphagic (Figure (Figure1D)1D) and had no detectable hypothalamic Pomc mRNA (Figure (Figure1E),1E), but NTS Pomc mRNA levels were reduced only to 44% ± 3% (n = 6) of wild-type levels. In contrast, body weight and daily food intake were normal in arcPomc+/– mice despite the fact that hypothalamic Pomc mRNA levels were 50% of those in their Pomc+/+ siblings (Figure (Figure1,1, C–E).
Excision of the neo cassette by a cre recombinase transgene at preimplantation embryonic stages (24) reestablished central Pomc expression, demonstrating that the phenotype is fully reversible (Figure (Figure2A).2A). Rescued arcPomcrcue/rcue mice showed normal hypothalamic Pomc mRNA levels: 90% ± 11% in males and 84% ± 13% in females (P = 0.29 and P = 0.21 respectively, t tests vs. Pomc+/+ mice). Consequently, germline-rescued mice exhibited normal body weight and normal daily food intake (Figure (Figure2,2, B and C).
To investigate whether postnatal recovery of neuronal Pomc expression could normalize body weight in mice that developed and grew through weaning in the absence of central POMC peptides, we obtained compound arcPomc–/–;Cre-ERT mice after successive crossing of arcPomc+/– mice with transgenic mice expressing a tamoxifen-inducible (TAM-inducible) cre recombinase (Cre-ERT; ref. 25). The Cre-ERT transgene driven by a ubiquitously active CAAG promoter was chosen for these studies because it produced TAM-dependent recombination of a loxP-STOP reporter allele in the majority of cells in virtually all tissues examined (25). The cellular specificity of Pomc reactivation by Cre-ERT is due to the endogenous Pomc regulatory elements in the knock-in arcPomc– allele. At P25, in juvenile arcPomc–/–:Cre-ERT mice that were still lean, TAM-induced rescue of Pomc expression completely prevented obesity in both sexes (Figure (Figure3A).3A). Conversely, arcPomc–/– mice treated with TAM and arcPomc–/–:Cre-ERT mice treated with vehicle grew significantly heavier than Pomc+/+ or Pomc+/+:Cre-ERT control mice (P < 0.001). TAM administration at P60 to obese arcPomc–/–:Cre-ERT mice resulted in rapid weight loss during the first 3 weeks and, following a plateau, resumption of weight gain at the same rate as that of Pomc+/+ littermates (Figure (Figure3B;3B; from week 12 to 24; repeated measures ANOVA [RMA], genotype × time effect, P > 0.05). Despite their improvement, male mice rescued at P60 remained heavier than Pomc+/+ controls (P < 0.01). To further study the ability of the energy balance homeostat to restore body weight in cases of extreme obesity, we rescued Pomc expression in P180 arcPomc–/–:Cre-ERT mice (Figure (Figure3,3, C and D). Notably, the overweight of female mice treated at P180 decreased dramatically from approximately 164% to 41% of Pomc+/+ controls compared with a more modest reduction from approximately 109% to 59% of controls in males (Figure (Figure3C).3C). Although P180-rescued mice showed significant weight loss, they remained more overweight than mice rescued at P60. Moreover, arcPomc–/–:Cre-ERT male mice rescued at P180, after reaching a nadir, regained weight faster than Pomc+/+ controls (RMA, genotype × time effect, P < 0.01). Together, these results show that Pomc expression can be restored eutopically in Arc neurons of weanling mice, leading to complete normalization of subsequent growth and body weight. However, the improvement in body weight is progressively attenuated, as the age of Pomc rescue is delayed from weaning to adulthood.
The age and sex differences in treatment results cannot be simply attributed to variation in recombination efficiency of the mutant Pomc alleles, since the percentage and rostro-caudal distribution of immunoreactive arcuate POMC neurons was similar among all groups (~75%) after TAM (Supplemental Figure 1 and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI62543DS1). However, there was a trend toward decreased hypothalamic Pomc mRNA levels as the age of TAM treatment was delayed (Supplemental Table 1). An inverse correlation between hypothalamic Pomc mRNA and obesity scores could be the result of increased leptin resistance to signaling mechanisms that normally promote Pomc expression (26).
To further test our hypothesis that the preceding development of obesity was directly responsible for the attenuated phenotypic response to Pomc restoration at P60 compared with P25 in male mice, we studied additional cohorts of arcPomc–/– and arcPomc–/–:Cre-ERT mice whose body weight was matched to that of wild-type controls by food restriction from weaning until P60 (Figure (Figure3E).3E). After TAM treatment, the mice had free access to chow. The arcPomc–/–:Cre-ERT mice that had been prevented from becoming overweight by restriction of their food intake continued to have completely normal body weight growth and ad libitum food intake relative to controls for the 10 weeks after Pomc rescue. Conversely, arcPomc–/– mice reverted to a hyperphagic state 2 weeks after TAM treatment, with accelerated weight gain similar in trajectory to that of naive mutant mice. Hypothalamic Pomc mRNA levels in the rescued arcPomc–/–:Cre-ERT mice were 47% ± 10% (n = 3) of the levels in the corresponding lean wild-type mice. These results demonstrate that the body weight, but not age, at the time of arcuate Pomc rescue conditions the levels of food intake that will maintain subsequent body weight at differential set points, as observed when comparing ad libitum fed versus food-restricted P60 Pomc-rescued mice (Figure (Figure3,3, B, left panel, and E).
ArcPomc–/–:Cre-ERT females, regardless of treatment age, completely normalized their food intake, as did males treated with TAM at P25 (Figure (Figure4A).4A). Although previously obese P60- and P180-rescued males also greatly reduced their food intake, they still exhibited a significant residual hyperphagia (12%–20% greater than controls; Figure Figure4,4, A and B). Food intake fluctuations paralleled the alterations in body weight, since all Pomc-rescued mice were profoundly hypophagic during the first 2–3 weeks after TAM administration (Figure (Figure4,4, B and C), causing them to lose weight during the same period (see Figure Figure3,3, B and C, and Figure Figure44C).
Since persistent overweight despite normalization of food intake might be attributed to decreased energy expenditure, a separate cohort of P60 mice was examined by indirect calorimetry before and after Pomc rescue. Body composition measured by NMR prior to TAM treatment demonstrated that the increased body weight of arcPomc–/– mice was primarily due to excessive fat; however lean body mass was also significantly increased (OWA F[2,17] = 21.3, P < 0.0001 females; F[2,15] = 8.3, P < 0.001 males), consistent with their approximately 5% increased body length (data not shown) (Figure (Figure5A5A and Supplemental Figure 2A). However, the weight loss induced by Pomc recovery in arcPomc–/–:Cre-ERT mice was solely due to reductions in fat but not lean mass. Obese mice of both sexes showed reduced locomotor activity before treatment that was normalized after Pomc rescue (Figure (Figure5B5B and Supplemental Figure 2B). In contrast, obese mice showed an approximately 10% reduction in oxygen consumption corrected for lean body mass that was unchanged after Pomc rescue (Figure (Figure5C5C and Supplemental Figure 2C). Together, these results suggest that the body weight improvement in rescued arcPomc–/–:Cre-ERT mice is determined by the restoration of melanocortin anorectic activity that follows the recovery of Pomc expression. The residual overweight of male arcPomc–/–:Cre-ERT mice rescued at P60 and P180 may have been caused by a small but persistent degree of hyperphagia, while female P60- and P180-rescued mice probably could not completely normalize their body weight because of persistently reduced energy expenditure.
Secondary conditions, such as ectopic fat deposition and diabetes, are major comorbidities in obesity. Therefore, we evaluated the improvement of several metabolic parameters in hypothalamic Pomc-rescued mice. Visceral (gonadal and retroperitoneal) and subcutaneous (inguinal) fat stores were normalized in all groups of TAM-treated arcPomc–/–:Cre-ERT mice, with the exception of inguinal fat pads in P180-rescued males (Supplemental Figure 3, A and B). Importantly, arcPomc–/–:Cre-ERT rescued mice of all groups showed complete normalization of liver weight due to a remarkable reduction in steatosis (Supplemental Figure 3, A and B). To determine whether weight loss in mice treated at P180 was accompanied by metabolic benefits, despite an incomplete normalization of body weight, we measured serum glucose, insulin, and leptin and calculated the homeostatic model assessment (HOMA) index of insulin resistance before and after Pomc rescue. As shown in Table Table1,1, those parameters greatly improved in arcPomc–/–:Cre-ERT mice after body weight loss.
Finally, we investigated food intake responses to starvation in arcPomc–/–:Cre-ERT mice treated at P180 with TAM. After a 24-hour fast, female arcPomc–/– mice failed to exhibit the normal fast-induced hyperphagia triggered in Pomc+/+ mice, while male arcPomc–/– showed a significantly reduced response (Figure (Figure66 and Supplemental Figure 4). Conversely, TAM-treated arcPomc–/–:Cre-ERT mice of both sexes recovered the fast-induced hyperphagic response. The absolute food intake of Pomc+/+ and rescued arcPomc–/–:Cre-ERT mice during the first 24 hours of refeeding matched the pre-fasting food intake of obese arcPomc–/– mice (Supplemental Figure 4). Overall, these results suggest that the lack of central POMC peptides caused mice to eat as if they were under continuous starvation and, therefore, insensitive to actual food deprivation. Since TAM-treated arcPomc–/–:Cre-ERT mice recovered hyperphagia induced by fasting, we conclude that the dynamic role of arcuate POMC neurons as integrators of energy balance circuits is restored in these mice.
By inserting a loxP-flanked neo cassette into the neuronal enhancer module of Pomc, we produced a mouse strain carrying a reversible genetic deficit that allowed us to directly measure the elastic limit of body weight regulation. A major advantage of this novel mouse model is that early-onset severe obesity is caused by inhibiting the expression of a single gene (Pomc), in a specific population of arcuate neurons, which can be reactivated by TAM-induced removal of the interfering neo cassette. An additional advantage of this model of extreme hyperphagia and obesity is that mice only have access to regular chow with low hedonic value, which precludes the sensitization of reward circuits normally activated by highly palatable food used in diet-induced obesity paradigms.
Pomc expression rescue in Arc neurons of weanling mice resulted in complete normalization of subsequent body weight. We ascribe the phenotypic rescue specifically to Arc neurons because of the small number of NTS POMC neurons, the very low basal levels of Pomc mRNA in the NTS relative to Arc, and the demonstration that obese arcPomc–/– mice retained a substantial fraction of NTS Pomc expression prior to TAM treatment. Our results have several important implications regarding the central POMC system. First, despite the precocious differentiation of POMC neurons in the embryonic hypothalamus (27), POMC peptides are not required during prenatal brain development or in the first 3 weeks of postnatal life to establish a normal body weight set point. Moreover, our results demonstrate that POMC neurons are normally specified during development and properly incorporated into functional neural circuits in the complete absence of POMC peptides, as indicated by the expression of a POMC-EGFP transgene mimicking the expression pattern of Pomc in hypothalamic neurons and the functional rescue of Pomc expression in the adult Arc. The physiological role of hypothalamic POMC prior to P25 remains to be fully defined.
The improvement in body weight of arcPomc–/– mice was progressively attenuated as the rescue of Pomc expression was delayed after weaning, despite the fact that TAM/CreER-mediated somatic recombination of the floxed-neo Pomc alleles was similar for both sexes and for all ages and body weights at the time of treatment. This finding supports the conclusions drawn from both clinical (6, 7) and rodent models (11–13), which relied on high-fat diet–induced obesity, that a condition of chronic overweight leads to secondary adaptations in the central regulation of energy homeostasis that act to perpetuate obesity despite reductions in caloric intake. Furthermore, since we controlled for treatment, genetic, and environmental factors by rescuing central Pomc expression in inbred mouse siblings fed a standard chow of low hedonic value, our results show for the first time to our knowledge that obesity is a primary condition that permanently alters the normal body weight set point by imposing a maladaptive allostatic state that ultimately defends a greater body weight. This conclusion is supported by our demonstration that prevention of excessive weight gain by calorie restriction in arcPomc–/–:CreERT mice extended the time window for complete normalization of subsequent body weight by Pomc rescue. Although the final body weight reached by extremely obese mice after treatment was still 40%–60% greater than that of normal lean mice, the reduction in weight was sufficient to significantly reduce comorbidities associated with obesity. This phenomenon is usually seen in severely obese patients who lose approximately 10% or more of their weight (28). However, it should be noted that the improvement in glucose homeostasis could also be due to weight loss–independent actions of the restored melanocortin peptides in the brain. The activation of MC4 receptors selectively reexpressed on cholinergic sympathetic preganglionic neurons has been demonstrated to be sufficient to attenuate hyperglycemia and hyperinsulinemia without affecting food intake or adiposity (29).
The two notable parameters associated with weight loss after the induction of hypothalamic Pomc were reduction in food consumption and normalization of locomotor activity. The immediate phase of extreme hypophagia lasting for 2–3 weeks after induction of Pomc expression conceivably was due to MCR supersensitivity resulting from the previous lack of melanocortin signaling (30). Energy expenditure, as measured by oxygen consumption, was not altered after Pomc rescue in either sex and actually remained significantly decreased in female mice, despite their increased locomotor activity. It is conceivable that a reduction in diet-induced thermogenesis accompanying the decreased food intake of the rescued mice obscured the expected increase in energy expenditure from the increased locomotion. However, our study was not designed to accurately discriminate between these two components of thermogenesis and basal metabolic rate. Recent reviews have also emphasized the complexity of relying on indirect calorimetry to evaluate thermogenesis in mutant mice that vary significantly in body composition from control animals or over time, as in the present study (31, 32). Therefore, the role played by hypothalamic POMC in the regulation of food intake was clearly restored after Pomc rescue, while its role in the control of energy expenditure was apparently resistant to treatment. This could be a consequence of different pathways mediating both roles of melanocortin peptides through MC4 receptors (29, 33). Alternatively, the reduction in oxygen consumption could be an indirect consequence of obesity rather than a direct effect of Pomc deficiency (34). In either case, the drop in energy expenditure contributes to the inability to restore a normal body weight set point.
The loss and restoration of β-endorphin, in parallel with those of the melanocortin peptides, likely also play a role in the body weight phenotype of these mice. We previously showed that mice with a selective absence of β-endorphin develop a mild obesity phenotype, particularly in males (35). Furthermore, a recent study suggested that an endogenous opioid, likely β-endorphin released from POMC neurons, inhibits hypothalamic AgRP neurons (36). Therefore, the absence of β-endorphin in arcPomc–/– mice may lead to a disinhibition of AgRP neurons that would complement the absence of melanocortin peptides and combine to induce severe hyperphagia.
Obese arcPomc–/– mice were hyperleptinemic before treatment, a condition probably leading to partial leptin resistance (37). There was a direct correlation between serum leptin levels before treatment (not shown for mice treated at P60), the weight at the time of Pomc rescue, the extent of hypophagia after treatment, and the percentage of weight loss. Furthermore, the restoration of fast-induced overfeeding suggests the reestablishment of Pomc responses to leptin in rescued arcPomc–/–:Cre-ERT mice, since this adaptation has been shown to be mediated, at least in part, by leptin activation of Stat3 (38). On the other hand, we found an inverse correlation between weight at the time of treatment and the subsequent levels of hypothalamic Pomc mRNA. Mice rescued at P25, which were lean at the moment of treatment and presumably normoleptinemic, recovered more than 50% of Pomc mRNA levels and defended a normal body weight. In contrast, mice rescued at P60 or P180 after the development of obesity and hyperleptinemia had attenuated Pomc expression below the threshold capable of sustaining normal satiety (<50%), consistent with a secondary phenotypic influence of leptin resistance, and therefore defended a higher body weight set point. This and other mechanisms such as the failure to normalize energy expenditure would contribute to the progressive waning of body weight normalization associated with treatment delay.
A recurring aspect of our study was the sexual dimorphism in treatment responses. Female mice had greater improvement in food intake and body weight. Both humans and rodents show sex-associated differences in fat distribution and leptin secretion (39). As females are more sensitive to leptin than males (40), it is possible that the hypophagia and body weight loss elicited by POMC immediately after rescue are influenced by leptin action (through POMC cells), although with greater response in females. It is likely that estrogen’s anorectic effect is also responsible for this dimorphism, since it is known that i.c.v. estradiol injection induces anorexia and increases excitatory synapses on POMC cells (41). In addition, we previously found that estrogen receptor α (ESR1) is a candidate nuclear receptor factor to regulate neuronal POMC expression, since it is expressed in POMC neurons during development and adulthood (42). Finally, although not evaluated in the present study, it is also possible that alterations in the posttranslational processing of POMC to its bioactive peptides in association with sex, aging, and preceding obesity status contribute to the allostatic state following the restoration of Pomc expression.
In conclusion, our findings show that obesity is a self-perpetuating condition and reinforce the importance of early consultation and weight management in children to prevent obesity, especially when taking into account that the probability of adult obesity exceeds 50% in people who were overweight at 6 years of age (43).
Mice were housed in ventilated cages under controlled temperature and photoperiod (12-hour light/ 12-hour dark cycle, lights on from 6 a.m. to 6 p.m.), with tap water and laboratory chow containing 28.0 kcal% protein, 12.1 kcal% fat, and 59.8 kcal% carbohydrate available ad libitum.
Mice carrying a floxed neo-cassette inserted in the neuronal Pomc enhancer neighborhood were generated as follows: The targeting vector was constructed with genomic sequences between –13 and –6.5 kb of mouse Pomc that were previously isolated (19) and a transgene construct harboring a 172-bp deletion of Pomc neuronal enhancer nPE2 (18). A neomycin-resistance cassette (PGK-neo) flanked by loxP sites was inserted 40 bp upstream of the deleted nPE2 region (10.3 kb upstream of the Pomc transcriptional start site). See Supplemental Methods for more details.
ArcPomc+/– mice were crossed with Cre-ERT mice (25) (B6.Cg-Tg[cre/Esr1]5Amc/J; The Jackson Laboratory), previously backcrossed with C57BL/6J for 5 generations, to obtain arcPomc+/–:Cre-ERT mice. Thereafter, arcPomc+/–:Cre-ERT were mated with arcPomc+/– mice to obtain all control and treated groups.
Three cohorts of mice were generated by breeding strategies described above and treated at different ages: postnatal days 25, 60, or 180 ± 3 days (P25, P60, and P180, respectively). A fourth cohort of male arcPomc–/– and arcPomc–/–:Cre-ERT mice was subjected to food restriction from the time of weaning to match body weights of Pomc+/+ and Pomc+/+:Cre-ERT lean control mice. After TAM treatment at P60, they were allowed to consume food ad libitum. All mice were individually housed within the first week after weaning and weighed twice a week at 3 p.m. At the indicated age, mice were treated either with 5 consecutive daily i.p. injections of 50 mg/kg/d TAM (Sigma-Aldrich) prepared as described previously (44) or with the oil vehicle as control. Food intake was calculated by weighing food from cages every day from Monday through Friday (3 p.m.), from 2 weeks before treatment (P60 and P180) until the fourth week after treatment (P25, P60, P180), and during an additional 2 weeks before ending experiments. Blood samples were collected 1 week before (P60, P180) treatment and 1 week before ending experiments (P25, P60, P180). Six weeks after treatment, P180-treated mice were subjected to a 24-hour fast (9 a.m.–9 a.m.) and then were allowed to eat ad libitum, and food was weighed after 0, 2, 8, 24, 48, and 72 hours during the refeeding period. Daily food intake during the previous week was used as a reference. Most of the mice were killed by cervical dislocation or decapitation at 5–6 months of age (P25 and P60) or 8–9 months of age (P180), individual fat pads and other organs were weighed, and the hypothalamus was snap frozen for later RNA extraction. The remaining mice were anesthetized and perfused with 4% paraformaldehyde for subsequent immunohistochemistry.
A fifth cohort of P60 arcPomc–/–:Cre-ERT:POMC-EGFP mice was generated for a repeated measures study of energy expenditure by indirect calorimetry (VO2, VCO2) and locomotor activity using a Comprehensive Lab Animal Monitoring System (CLAMS) (Columbus Instruments). These mice were individually housed starting at 8 weeks of age and treated with TAM as above at P70 ± 6 days. Data were collected from the mice in the CLAMS units for 3 days immediately before TAM treatment and for a second 3-day period, 5 weeks later. Oxygen consumption (VO2) was corrected for lean body mass determined by body composition analysis using NMR (Minispec LF9011, Bruker) immediately after each CLAMS session.
Vibratome sections (50 μm) of paraformaldehyde-perfused brains were subjected to immunohistochemistry with an anti–rat ACTH antibody (1:10,000, A.F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, California, USA) and developed with diaminobenzidine (DAB; Sigma-Aldrich) or detected by Alexa Fluor 555–coupled anti-rabbit IgG (Molecular Probes, Invitrogen). Images were taken with an Olympus DP72 digital camera connected to an upright research microscope (Olympus BX51). The number of DAB-stained POMC neurons (positive for ACTH) in rescued mice was estimated all across the Arc and matched with equivalent slices of TAM-treated Pomc+/+ mice to calculate percentage of recovery. For immunofluorescence, ACTH-positive cells (red) were colocalized with endogenous EGFP fluorescence (green). E14 sections were developed with SG staining (Vector). See Supplemental Methods for more details.
All data presented are the mean ± SEM and were analyzed by RMA or OWA unless otherwise stated using Statistica (version 7). Post hoc pairwise comparisons between groups were performed by Bonferroni’s post hoc test. P values less than 0.05 were considered significant.
All procedures were approved by the Institutional Animal Care and Use Committees and followed the Public Health Service guidelines for the humane care and use of experimental animals.
We thank R. Kruse Bend, B. Warren, and V. Rodríguez for valuable technical support and N. Qi for analysis of calorimetry data. This work was supported by NIH grants DK066604 (to M.J. Low) and DK068400 (to M.J. Low and M. Rubinstein); International Research Scholar Grant of the Howard Hughes Medical Institute (HHMI) (to M. Rubinstein); Agencia Nacional de Promoción Científica y Tecnológica, Argentina (to V.F. Bumaschny and M. Rubinstein); Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (to V.F. Bumaschny, F.S.J. de Souza, and M. Rubinstein); and Universidad de Buenos Aires. This work utilized core services provided by the Oregon Health and Science University Transgenic Core Laboratory and the University of Michigan Animal Phenotyping Core supported by the Michigan Diabetes Research and Training Center and the Michigan Nutrition and Obesity Research Center (NIH grants DK020572 and DK089503).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(11):4203–4212. doi:10.1172/JCI62543.
See the related Commentary beginning on page 3840.