Long-term consequences of the absence of leptin signaling in early life
The hormone leptin is best known as a key signal to the brain conveying information about body energy reserves ( Maffei et al . , 1995 ) . Consequently , leptin receptor ( LepR ) -expressing neurons are apt to modulate food intake and energy expenditure according to the amount of energy stored and the current energy balance ( Ramos-Lobo and Donato , 2017 ) . Hence , null mutations in the genes that encode for leptin or LepR impair the capacity of the hypothalamus to regulate energy homeostasis , leading to profound metabolic dysfunctions in humans and rodents , including severe obesity , hyperphagia , low-energy expenditure , hypoactivity , insulin resistance and infertility ( Halaas et al . , 1995; Montague et al . , 1997; Farooqi et al . , 2002; Licinio et al . , 2004 ) . In addition to the role of leptin in energy homeostasis , other studies have shown that leptin can affect developmental processes , especially in the brain ( Ahima et al . , 1999 ) . Regarding this aspect , rodents exhibit a postnatal leptin surge between the first and second week of age that is not associated to changes in fat mass or food intake ( Ahima et al . , 1998 ) . In addition , 2-week-old mice do not respond to the anorexigenic effects of leptin ( Mistry et al . , 1999 ) . Thus , leptin does not seem to be involved in the regulation of energy balance during the first weeks of life in mice . However , synaptic plasticity in the hypothalamus is regulated by leptin ( Pinto et al . , 2004 ) . Leptin signaling is also required for the formation of the neural projections from the arcuate nucleus ( ARH ) to post-synaptic targets , a process that takes place in the first 2 weeks of life ( Bouret et al . , 2004a; Kamitakahara et al . , 2018 ) . In accordance , leptin promotes neurite outgrowth in vitro and leptin-deficient ( Lepob/ob ) mice show disruption of ARH neural projections ( Bouret et al . , 2004b ) . Importantly , exogenous leptin rescues the development of ARH projections of Lepob/ob mice only when provided in the neonatal period . Thus , these findings suggest that the lack of leptin signaling in the first weeks of life permanently disrupts these projections in mice ( Bouret et al . , 2004b ) . Important implications arise from the developmental effects of leptin . For example , changes in leptin signaling during development could then affect the organization of critical neural circuits that regulate energy homeostasis , favoring the incidence of metabolic diseases later in life . Thus , leptin may also be involved in the developmental programming ( Ralevski and Horvath , 2015 ) . Accordingly , early postnatal leptin blockage in rats leads to long-term leptin resistance and susceptibility to diet-induced obesity ( Attig et al . , 2008 ) . Furthermore , neonatal leptin treatment reverses the developmental programming caused by maternal undernutrition ( Vickers et al . , 2005 ) . However , it is worth mentioning that despite the aforementioned evidence about leptin’s developmental effects , exogenous leptin replacement can reverse key metabolic abnormalities exhibited by leptin-deficient mice or humans ( Pelleymounter et al . , 1995; Ioffe et al . , 1998; Farooqi et al . , 2002; Licinio et al . , 2004; Donato et al . , 2011 ) . Therefore , the actual importance of leptin signaling during development for the long-term energy homeostasis and other aspects regulated by leptin is not yet fully understood . Thus , to uncover the role of leptin during development , we investigated the long-term consequences of the absence of leptin signaling in early life . For this purpose , we studied knockout mice for the Lepr gene that grew without leptin’s effects . However , LepR expression was restored either at the fourth or tenth week of life . Our findings revealed that some defects previously considered irreversible due to the lack of leptin signaling in the neonatal period , such as the poor development of ARH neural projections , can be recovered by LepR reactivation in adulthood . On the other hand , the absence of leptin signaling in early life permanently impairs energy homeostasis , the melanocortin system , as well as the reproduction and brain development .
To study the importance of leptin signaling during development , we used the LepRNull mouse model that carries a transcription blocker between exons 16 and 17 of the Lepr gene ( Berglund et al . , 2012 ) , preventing the expression of the long LepR isoform ( Lepr-b ) . As expected , adult male and female LepRNull mice were morbidly obese and hyperphagic ( Figure 1A–D ) . Since the transcription blocker of LepRNull mice is flanked by LoxP sites , a temporal Cre expression can permanently remove this cassette . LepRNull mice were bred with animals expressing Cre-ERT2 fusion protein under the human ubiquitin C promoter sequence . Thus , tamoxifen injections were able to induce Cre recombinase activity ubiquitously , restoring LepR expression in Ubi-LepRNull mice . We decided to induce LepR reactivation when mice were 10 weeks old because the vast majority of developmental processes were completed at this age . We observed that tamoxifen injections caused no long-term changes in body weight or food intake in lean controls ( Ubi group ) and LepRNull mice ( Figure 1A–D ) . In contrast , Ubi-LepRNull mice exhibited a sustained weight loss and marked food intake reduction in the first 4 weeks after tamoxifen treatment ( Figure 1A–D ) . To confirm the gene reactivation , we assessed the hypothalamic expression of Lepr-b mRNA and observed the expected suppression in LepRNull mice ( 0 . 01 ± 0 . 00 a . u . ; n = 8 ) , compared to lean controls ( 1 . 00 ± 0 . 06 a . u . ; n = 8 ) . Importantly , LepR reactivation recovered the expression of Lepr-b mRNA in Ubi-LepRNull mice ( 0 . 66 ± 0 . 04 a . u . ; n = 8; p=0 . 071 vs . Ubi mice ) . To further validate our experimental model , we evaluated the leptin responsiveness in key hypothalamic nuclei that contain LepR-expressing neurons ( Ramos-Lobo and Donato , 2017 ) . For this purpose , we assessed the capacity of leptin to induce the phosphorylation of STAT3 ( pSTAT3 ) , which depends on the long LepR isoform ( Bates et al . , 2003 ) . As expected , an i . p . leptin injection induced a robust pSTAT3 immunoreactivity in the ventromedial subdivision of the ARH ( ARHvm ) , lateral subdivision of the ARH ( ARHl ) , dorsomedial nucleus ( DMH ) , ventromedial nucleus ( VMH ) and ventral premammillary nucleus ( PMv ) of control Ubi mice ( Figure 1E , H ) . In accordance with the deficiency of the long LepR isoform , LepRNull mice show virtually no pSTAT3 in the hypothalamus ( Figure 1F , I ) . Importantly , tamoxifen injections restored leptin responsiveness in Ubi-LepRNull mice to a similar extent as that found in the Ubi group ( Figure 1G , J , K ) . To functionally assess leptin sensitivity , we determined the acute anorexigenic effects caused by an i . p . leptin injection . Leptin induced a similar decrease of food intake and body weight in Ubi and Ubi-LepRNull mice , whereas LepRNull mice remained unresponsive to leptin ( Figure 1L ) . Altogether , these findings demonstrate that although mice were depleted from the effects of leptin until the 10th week of life , tamoxifen injections completely restored leptin signaling in adult Ubi-LepRNull mice . We found that body weight and food intake of Ubi-LepRNull mice were stabilized 6 weeks after tamoxifen treatment ( Figure 1A–D ) . Therefore , all remaining analyses were performed after this period . Despite the marked weight loss induced by LepR reactivation , Ubi-LepRNull male mice remained heavier and had higher body adiposity and leptinemia , compared to control Ubi mice ( Figure 2A–C ) . These changes occurred despite a complete normalization of food intake in Ubi-LepRNull mice ( Figure 2D ) . On the other hand , oxygen consumption ( VO2 ) remained significantly lower in Ubi-LepRNull male mice compared to lean controls ( Figure 2E , F ) , indicating that the increased adiposity of Ubi-LepRNull mice could be explained by a partial recovery in their energy expenditure . The respiratory exchange ratio ( RER ) of Ubi-LepRNull male mice was normalized , restoring the circadian rhythm of fuel oxidation which was absent in LepR deficient mice ( Figure 2G , H ) . As seen in the energy expenditure , the phenotype of hypoactivity observed in LepRNull mice was only partially restored in Ubi-LepRNull male animals ( Figure 2I , J ) . The energy homeostasis was also evaluated in females . As seen in males , Ubi-LepRNull females remained heavier and had higher body adiposity compared to Ubi mice ( Figure 2—figure supplement 1A , B ) , although serum leptin levels and food intake were completely normalized ( Figure 2—figure supplement 1C , D ) . Ubi-LepRNull females also exhibited a partial recovery in their energy expenditure in the dark phase compared to Ubi mice ( Figure 2—figure supplement 1E , F ) . Additionally , RER was normalized after LepR reactivation ( Figure 2—figure supplement 1G , H ) , whereas Ubi-LepRNull females displayed a partial recovery in their ambulatory activity , compared to Ubi mice ( Figure 2—figure supplement 1I , J ) . Food deprivation produces compensatory decreases in energy expenditure as a defense mechanism against weight loss ( Leibel et al . , 1995 ) . Thus , we subjected the animals to 24 hr fasting to evaluate their metabolic responses during a situation of negative energy balance ( Figure 3A–C ) . LepRNull and Ubi-LepRNull mice showed a lower fasting-induced weight loss compared to Ubi mice ( Figure 3A ) . Notably , while Ubi mice adapted to fasting by decreasing the energy expenditure , both LepRNull and Ubi-LepRNull mice exhibited an attenuated reduction compared to baseline ( Figure 3B , C ) . Overfeeding can also produce compensatory responses in humans and mice in order to prevent excessive weight gain ( Leibel et al . , 1995 ) . We measured the food intake 4 , 12 , 24 and 48 hr after a 24 hr fasting period and observed a similar refeeding response between male and female Ubi and Ubi-LepRNull mice , whereas male LepRNull mice exhibited an increase in food intake after 24 and 48 hr of refeeding ( Figure 3—figure supplement 1A , B ) . Additionally , we evaluated the metabolic responses produced by a high-fat diet ( HFD ) intake for 48 hr . Interestingly , both LepRNull and Ubi-LepRNull mice showed higher HFD intake compared to Ubi animals ( Figure 3D ) . However , HFD-induced thermogenesis was similar among the experimental groups ( Figure 3E , F ) . To investigate the possible causes of the metabolic imbalances exhibited by Ubi-LepRNull mice , we assessed the hypothalamic expression of key genes involved in energy balance regulation . As expected , LepRNull mice showed increased agouti-related protein ( Agrp ) and neuropeptide Y ( Npy ) mRNA levels in the hypothalamus , whereas the expression of proopiomelanocortin ( Pomc ) and cocaine and amphetamine regulated transcript ( Cartpt ) was reduced compared to Ubi mice ( Figure 3G ) . LepR reactivation downregulated Agrp and Npy mRNA levels in the hypothalamus of Ubi-LepRNull mice . However , Pomc and Cartpt expression was not restored and remained significantly lower compared to control Ubi mice ( Figure 3G ) . To assess whether the absence of leptin signaling in early life could have affected the viability of POMC neurons , we determined the number of α-melanocyte-stimulating hormone ( α-MSH ) or β-endorphin immunoreactive cells in the ARH ( Figure 3—figure supplement 2 ) . We observed a similar number of ARH POMC neurons between Ubi and Ubi-LepRNull mice ( Figure 3—figure supplement 2 ) . Neurons outside the ARH also regulate the thermogenic effect of leptin , including those that express the RFamide prolactin-releasing peptide ( Prlh ) in the DMH ( Dodd et al . , 2014 ) . Thus , we assessed the hypothalamic expression of Prlh mRNA , which was suppressed in LepRNull mice and remained lower in the hypothalamus of Ubi-LepRNull mice ( Figure 3G ) . Epigenetic changes in early life can cause long-term consequences in energy homeostasis and increase the risk of obesity in adulthood ( Lillycrop and Burdge , 2011 ) . Hence , the expression of some histone deacetylases ( Hdac ) was also determined and we observed that Hdac3 and Hdac5 mRNA levels were suppressed in the hypothalamus of LepRNull mice and were not normalized after LepR reactivation ( Figure 3G ) . Hdac8expression was increased in the hypothalamus of LepRNull mice , but it returned to normal values in Ubi-LepRNull mice ( Figure 3G ) . DNA methyltransferase ( Dnmt ) enzymes catalyze the transfer of a methyl group to DNA , affecting many biological functions including the energy homeostasis via hypothalamic neurons ( Kohno et al . , 2014 ) . We analyzed the expression of some Dnmt and we observed that Dnmt3a and Dnmt3b mRNA levels were suppressed in the hypothalamus of both LepRNull and Ubi-LepRNull mice , compared to Ubi animals ( Figure 3G ) . No changes among the groups were observed in the hypothalamic expression of Dnmt1 ( Figure 3G ) . Glucose homeostasis was also evaluated in our experimental animals . LepRNull mice showed the expected glucose intolerance , as well as increased insulin resistance and serum insulin concentrations ( Figure 4 ) . Glucose tolerance of Ubi-LepRNull mice was normalized in both males ( Figure 4A , B ) and females ( Figure 4F , G ) . However , Ubi-LepRNull mice remained less responsive to insulin ( Figure 4C , D , H , I ) . In accordance with their insulin resistance , serum insulin concentrations remained significantly higher in Ubi-LepRNull mice , compared to Ubi mice ( Figure 4E , J ) . Thus , absence of leptin signaling in early life produced long-term alterations in glucose homeostasis . LepR reactivation in 10-week-old mice induced a marked weight loss ( Figure 1A , C ) . Thus , it is unclear whether the alterations in the energy and glucose homeostasis of Ubi-LepRNull mice were caused by the absence of leptin in early life or were associated with the metabolic consequences of a robust weight loss ( Leibel et al . , 1995 ) . To answer this question , we produced a new group of animals in which tamoxifen treatment started at the fourth week of life ( Figure 5—figure supplement 1 ) . Therefore , LepR reactivation was induced before the onset of obesity , and consequently Ubi-LepRNull mice were not exposed to weight loss ( Figure 5—figure supplement 1 ) . Hypothalamic gene expression analysis indicated that Ubi-LepRNull mice exhibited a similar Lepr-b mRNA expression ( 1 . 26 ± 0 . 11 a . u . ; n = 7 ) compared to Ubi mice ( 1 . 00 ± 0 . 09 a . u . ; n = 8; p=0 . 5263 ) , whereas LepRNull mice showed suppressed hypothalamic Lepr-b mRNA levels ( 0 . 01 ± 0 . 00 a . u . ; n = 7 ) . We started the in vivo analyses at the same age of previously studied adult animals ( approximately 15 weeks of age ) . LepR reactivation before the onset of obesity did not affect the body weight of Ubi-LepRNull males in the long-term , but Ubi-LepRNull females still showed higher body weight than Ubi mice ( Figure 5A ) . Notably , both male and female Ubi-LepRNull mice exhibited increased fat deposition in the perigonadal depot ( Figure 5B ) , although no differences were observed in other fat pads ( data not shown ) or in serum leptin concentration in male mice ( Figure 5C ) . Food intake was also completely normalized in male and female Ubi-LepRNull mice ( Figure 5D ) . As seen in adult animals , LepR reactivation before the onset of obesity led to suppressed energy expenditure in the dark phase and over a 24 hr period ( Figure 5E , F ) . In contrast , RER and ambulatory activity were normalized in young Ubi-LepRNull male mice ( Figure 5G–J ) . Glucose homeostasis was also evaluated in mice that received tamoxifen treatment before the onset of obesity . In contrast to the results observed after LepR reactivation in adult mice , LepR reactivation before the onset of obesity completely normalized the glucose tolerance and insulin sensitivity in both male and female Ubi-LepRNull mice ( Figure 5—figure supplement 2 ) . Leptin not only controls the energy and glucose homeostasis , but it also plays an important role regulating several endocrine systems ( Ahima et al . , 1996; Ramos-Lobo and Donato , 2017 ) . Thus , we also evaluated the endocrine consequences of the absence of leptin signaling in early life . We initially assessed the thyroid axis and observed that LepRNull males exhibited the expected suppression in serum T4 concentration ( Ahima et al . , 1996 ) , while Ubi-LepRNull mice showed similar values to Ubi animals ( Figure 6A ) . LepRNull mice also displayed higher serum corticosterone concentration compared to Ubi animals , whereas LepR reactivation normalized the hypothalamic-pituitary-adrenal axis ( Figure 6B ) . Next , the reproductive system was evaluated . In males , LepRNull mice showed lower serum testosterone concentration compared to Ubi animals , whereas Ubi-LepRNull mice exhibited intermediate values ( Figure 6C ) . Notably , LepR reactivation in adult animals was unable to completely restore testicle weight , which was reduced by LepR deficiency ( Figure 6D ) . To evaluate how these changes affected their fertility , Ubi and Ubi-LepRNull males were mated with wild-type females for 28 days . We observed that only 10% ( 1 out 10 ) of females mating with Ubi-LepRNull males got pregnant , whereas the percentage of pregnancy increased to approximately 50% when females mated with Ubi mice . Leptin deficiency impairs hypothalamic gonadotropin-releasing hormone ( GnRH ) release , leading to hypogonadotropic hypogonadism ( Chehab et al . , 1996; Licinio et al . , 2004 ) . Consequently , GnRH peptide content accumulates in the median eminence/mediobasal hypothalamus of food-deprived or LepR-deficient animals ( Polkowska et al . , 2006; Donato et al . , 2011 ) . We evaluated the density of GnRH fibers reaching the median eminence and observed the expected increase in the integrated optical density of GnRH staining in LepRNull mice , compared to Ubi animals ( Figure 6E , F ) . Remarkably , LepR reactivation did not restore GnRH fiber density in the hypothalamus of Ubi-LepRNull mice ( Figure 6G , H ) . GnRH release is stimulated by several neurotransmitters , including the kisspeptins ( Messager et al . , 2005 ) and nitric oxide ( Bellefontaine et al . , 2014 ) . Thus , we evaluated the hypothalamic mRNA expression of Kiss1 and Nos1 . We observed a reduction in the expression of these transcripts in the hypothalamus of LepRNull and Ubi-LepRNull mice ( Figure 6I ) , indicating that LepR reactivation in adults was not able to normalize Kiss1 and Nos1 mRNA levels . We only evaluated the testicle weight in mice that had the LepR reactivation before the onset of obesity . However , as seen in adult animals , LepR reactivation in the fourth week of life was not capable of restoring testicle weight to values found in Ubi mice ( Figure 6J ) . In females , the reproductive system was evaluated through the daily inspection of the vaginal smear . Ubi mice showed the expected variations in the estrous cycle ( Figure 6K ) . In contrast , the vaginal cytology of LepRNull mice indicated a prevalence of leukocytes and a complete absence of cornification and estrous cyclicity . LepR reactivation increased the appearance of cornified cells in the vaginal smear , although the disruption of the estrous cycle was still evident in Ubi-LepRNull mice ( Figure 6K ) . Thus , these data suggest disruption in the reproductive system of male and female mice that grew without leptin signaling until adulthood . Leptin or LepR deficiency leads to reduced brain mass ( Ahima et al . , 1999 ) , disruption of the projections from ARH neurons to the paraventricular nucleus of the hypothalamus ( PVH ) ( Bouret et al . , 2004a; Bouret et al . , 2004b; Kamitakahara et al . , 2018 ) , as well as behavioral and cognitive problems ( Liu et al . , 2011 ) . Therefore , we evaluated the possible consequences of the lack of leptin signaling in early life on brain development . LepRNull male and female mice exhibited reduced brain mass ( Figure 7A , B ) . Notably , LepR reactivation in adult animals partially recovered brain mass , although it remained significantly lower in Ubi-LepRNull mice compared to Ubi animals ( Figure 7A , B ) . We then assessed the hypothalamic expression of several neurotrophic factors . LepRNull mice exhibited reduced mRNA levels of Bdnf , Igf1 and Dlg4 , which encode important neurotrophic factors and proteins involved in synaptic plasticity and memory formation ( Figure 7C ) . The expression of none of these transcripts was completely restored in Ubi-LepRNull mice ( Figure 7C ) . Additionally , the glial fibrillary acidic protein ( Gfap ) expression was suppressed in the hypothalamus of LepRNull and Ubi-LepRNull mice in comparison with Ubi animals ( Figure 7C ) . The brain mass was also determined in mice that recovered LepR expression before the onset of obesity . While Ubi-LepRNull males still exhibited reduced brain mass compared to Ubi mice ( Figure 7D ) , no significant difference was observed between Ubi and Ubi-LepRNull females ( Figure 7E ) . To determine whether the aforementioned alterations in Ubi-LepRNull mice could lead to behavioral or cognitive deficits , male mice were subjected to behavioral tests . In the open field , we observed that LepRNull mice showed reduced exploration , whereas LepR reactivation normalized this behavior ( Figure 7—figure supplement 1A ) . Next , spatial learning and memory were analyzed in the Barnes maze . In this case , LepRNull mice were not evaluated due to their decreased voluntary ambulatory activity . Along the training period , Ubi and Ubi-LepRNull mice reduced the primary latency ( Figure 7—figure supplement 1B; p<0 . 0001 ) and the number of primary errors ( Figure 7—figure supplement 1C; p<0 . 0001 ) to reach the escape box , indicating that both groups learned the task . During the test sessions , Ubi and Ubi-LepRNull mice also showed similar learning capacity since the time spent in the target zone were similar between the groups 24 hr ( Figure 7—figure supplement 1D , E ) and 1 week ( Figure 7—figure supplement 1F , G ) after the training period . Previous studies have shown that there is a critical neonatal period , in which leptin plays a neurotrophic role inducing the development of neural projections from ARH neurons to post-synaptic targets , including the PVH ( Bouret et al . , 2004b; Kamitakahara et al . , 2018 ) . To initially investigate the projections of ARH neurons to the PVH , we crossed Lepob/+ mice with LepR-Cre::LSL-tdTomato mice , in order to produce leptin deficient mice expressing the tdTomato fluorescent protein only in LepR cells . Thus , we compared the distribution of axons in the PVH between control ( LepR-Cre::LSL-tdTomato ) and Lepob/ob::LepR-Cre::LSL-tdTomato mice . We found a significant reduction in the density of axons from LepR-expressing neurons in the PVH of Lepob/ob mice compared to control animals ( Figure 7—figure supplement 2 ) . This result indicates that the absence of leptin signaling reduces the density of axons from LepR-expressing neurons that innervate the PVH . Next , we evaluated the density of AgRP and POMC fibers in the PVH to determine whether LepR reactivation in adults can restore these neural projections . We confirmed that LepRNull mice had reduced density of AgRP and POMC fibers in the PVH , compared to Ubi mice ( Figure 7F–M ) . However , in contrast to the findings of previous studies ( Bouret et al . , 2004b; Kamitakahara et al . , 2018 ) , our results indicate that restoration of leptin signaling in adults recovers the density of AgRP and POMC fibers in the PVH ( Figure 7F–M ) .
Our findings revealed that the absence of leptin signaling in early life led to permanent changes in energy homeostasis , melanocortin system , reproduction and brain development . The metabolic dysfunctions caused by the lack of leptin signaling during development possibly involved changes in neural populations that regulate the energy expenditure , such as POMC or Prlh neurons in the hypothalamus , as well as epigenetic mechanisms since the hypothalamic expression of several enzymes that modulate DNA methylation or histone acetylation were affected in Ubi-LepRNull mice . Although the complete absence of leptin signaling is rarely found in humans , both undernutrition and overnutrition are able to produce significant changes in serum leptin concentrations , leptin sensitivity or in the development of neurocircuits that regulate energy homeostasis ( Samuelsson et al . , 2008; Chen et al . , 2009; Vogt et al . , 2014; Ralevski and Horvath , 2015 ) . Therefore , intrauterine or early postnatal changes in energy balance have been consistently linked with obesity and other metabolic diseases in adulthood ( Vickers et al . , 2005; Attig et al . , 2008; Lillycrop and Burdge , 2011; Bumaschny et al . , 2012; Chhabra et al . , 2016 ) . Thus , our findings contribute to the understanding of the metabolic programming by indicating which specific metabolic , neuroendocrine and developmental consequences arise from the absence of leptin signaling in early life , especially in the context of the alarming growth of childhood obesity worldwide ( Cunningham et al . , 2014 ) .
Experiments were performed in male and female mice . To induce reactivation of the Lepr gene , a Lox-flanked transcription-blocking cassette ( loxTB ) was inserted in the Lepr gene to generate mice null for the Lepr ( STOCK Leprtm1Jke/J , The Jackson Laboratory ) . They were bred with animals expressing Cre-ERT2 fusion protein under the human ubiquitin C promoter sequence ( B6 . Cg-Ndor1Tg ( UBC-cre/ERT2 ) 1Ejb/2J , The Jackson Laboratory ) . As control animals , we used obese mice homozygous for the loxTB allele ( LepRNull group ) and lean control mice carrying only the Cre allele ( Ubi group ) . Mice carrying both the Cre transgene and the loxTB allele in homozygosity were considered the conditional knockout mice ( Ubi-LepRNull group ) . Mice in these strains were in the C57BL/6 background and all groups were composed of littermate animals . To visualize LepR-expressing cells , we generated a LepR-reporter mouse by breeding the LepR-Cre mouse ( B6 . 129-Leprtm2 ( cre ) Rck/J , The Jackson Laboratory ) with the lox-stop-lox ( LSL ) -tdTomato reporter mouse ( B6;129S6-Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) Hze/J , The Jackson Laboratory ) . Thus , axons from LepR-expressing cells were visualized without additional staining by the presence of the tdTomato fluorescent protein . We then crossed Lepob/+ mice ( B6 . Cg-Lepob/J , The Jackson Laboratory ) with LepR-Cre::LSL-tdTomato mice , in order to produce leptin deficient mice expressing the tdTomato fluorescent protein only in LepR cells . Mice were weaned at 3–4 weeks of age and their mutations were confirmed by genotyping the DNA that had been previously extracted from the tail tip ( REDExtract-N-Amp Tissue PCR Kit , Sigma ) . The genetically modified mouse models were produced and maintained in standard conditions of light ( 12 hr light/dark cycle ) and temperature ( 22 ± 1°C ) . Unless otherwise indicated , mice received a regular rodent low-fat chow diet ( 2 . 99 kcal/g; 9 . 4% calories from fat ) . All experiments were carried out in compliance with NIH guidelines for the care and use of laboratory animals and were previously approved by our Institutional Animal Ethics Committee ( protocol number 137/2013 ) . All mice received 5 i . p . injections of tamoxifen ( 0 . 15 mg/g , Sigma-Aldrich ) , diluted in sesame oil ( Sigma-Aldrich ) , in alternate days . Mice were treated with tamoxifen at 10 weeks of age ( adults; LepRNull and Ubi-LepRNull mice were already obese ) or at 4 weeks of age ( young; before the onset of obesity ) . The animals were monitored until stabilization of body weight and food intake . After the stabilization period , the in vivo experiments were carried out . At least 6 weeks after the last tamoxifen injection ( 16 weeks of age in adults or 15 weeks of age for young mice ) , mice were single housed . Their body weight and food intake were measured for 3 to 4 alternate days . Then , mice were subjected to a glucose tolerance test ( 2 g glucose/kg b . w . ; i . p . ) and to an insulin tolerance test ( 1 IU insulin/kg b . w . ; i . p . ) . To determine O2 consumption ( energy expenditure ) , CO2 production , respiratory exchange ratio ( RER ) and locomotor activity ( through infrared beam sensors ) mice were placed in the Oxymax/Comprehensive Lab Animal Monitoring System ( CLAMS; Columbus Instruments , Columbus , OH ) . After an adaptation period of 3 days inside the CLAMS , these metabolic parameters were evaluated for four consecutive days . Therefore , the results presented are the average of this period . Body adiposity was determined by weighting perigonadal ( PG ) , subcutaneous ( SC ) and retroperitoneal ( RP ) fat pads in males . For the females , the periovarian ( Ov ) fat pad was also included . To visualize leptin responsive cells in the brain , adult mice ( n = 3–4/group ) received an acute i . p . injection of mouse recombinant leptin ( 5 µg/g , from Dr . A . F . Parlow , National Hormone and Peptide Program – NHPP , National Institute of Diabetes and Digestive and Kidney Diseases ) and were perfused 90 min later . For the perfusions , mice were deeply anesthetized with isoflurane and perfused transcardially with saline , followed by a 10% buffered formalin solution ( 150–200 mL per mouse ) . Brains were collected and post-fixed in the same fixative for 60 min and cryoprotected overnight at 4°C in 0 . 1 M PBS containing 20% sucrose , pH 7 . 4 . Brains were cut ( 30 µm thick sections ) in the frontal plane using a freezing microtome . To label pSTAT3 , brain sections were rinsed in 0 . 02 M potassium PBS , pH 7 . 4 ( KPBS ) , followed by pretreatment in an alkaline ( pH >13 ) water solution containing 1% hydrogen peroxide and 1% sodium hydroxide for 20 min . After rinsing in KPBS , sections were incubated in 0 . 3% glycine and 0 . 03% lauryl sulfate for 10 min each . Next , sections were blocked in 3% normal donkey serum for 1 hr , followed by incubation in anti-pSTAT3Tyr705 primary antibody ( 1:1000; Cell Signaling; #9131 ) for 40 hr . AgRP , β-endorphin , α-MSH or GnRH immunoreactivity was evaluated in brain sections that were rinsed in KPBS and blocked in 3% normal donkey serum for 1 hr , followed incubation in anti-AgRP ( 1:2000 , Phoenix Pharmaceuticals; #H-003–53 ) , anti-β-endorphin ( 1:2000 , Phoenix Pharmaceuticals; #H-022–33 ) , anti-αMSH ( 1:2000 , Chemicon; #AB5087 ) or anti-GnRH ( 1:2000 , Immunostar; #AB_572248 ) primary antibodies . For the immunofluorescence reaction , sections were rinsed in KPBS and incubated for 90 min in AlexaFluor488 or AlexaFluor594-conjugated secondary antibodies ( 1:500 , Jackson Laboratories ) . Sections were mounted onto gelatin-coated slides and the slides were coverslipped with Fluoromount G ( Electron Microscopic Sciences , Hatfield , PA ) . For the immunoperoxidase staining , sections were incubated for 1 hr in biotin-conjugated secondary antibody ( 1:1000 , Jackson Laboratories ) and followed by 1 hr incubation with an avidin-biotin complex ( 1:500 , Vector Labs ) . The peroxidase reaction was performed using 0 . 05% 3 , 3'-diaminobenzidine , 0 . 25% nickel sulfate and 0 . 03% hydrogen peroxide resulting in a black nuclear staining . The slides were coverslipped with DPX mounting medium ( Sigma , St . Louis , MO ) . Brightfield and epifluorescence photomicrographs were acquired with a Zeiss Axiocam HRc camera coupled to a Zeiss Axioimager A1 microscope ( Zeiss , Munich , Germany ) . Images were digitized using Axiovision software ( Zeiss ) . Confocal images were captured in a 1024 × 1024 pixel format using a Zeiss LSM 780 confocal laser scanning inverted microscope ( Carl Zeiss , Germany ) . Image stacks comprised eight images captured with a LD Plan-Neofluar 40x/0 . 6 Korr M27 objective ( Zeiss ) . The ImageJ Cell Counter software ( http://rsb . info . nih . gov/ij/ ) was used to manually count the number of cells in the areas of interest . Fiber density was determined by measuring the integrated optic density using ImageJ software . To assess leptin sensitivity , adult mice received an i . p . injection of either phosphate-buffered saline ( PBS ) or mouse recombinant leptin ( 2 . 5 µg/g b . w . ; from Dr . A . F . Parlow , NHPP , USA ) 3 hr before their dark phase , and their food intake and body weight were recorded for 24 hr following the injection . Both parameters after PBS injection were compared with the values after leptin administration . To investigate the response to metabolic challenges of negative or positive energy balance , previously single-housed mice were initially subjected to a 24 hr fasting protocol . During this challenge , metabolic parameters were continuously assessed in the CLAMS , and their body weight was monitored at baseline and after 24 hr of fasting . Changes in metabolic parameters during fasting were reported as percentage of the values obtained from baseline ( fed state ) . Food intake was recorded 4 , 12 , 24 and 48 hr following the fasting period . After a recovery period ( at least 1 week ) , the animals were exposed to a HFD ( 5 . 31 kcal/g , 58% calories from fat ) for 48 hr . Metabolic parameters were assessed in the CLAMS , together with their body weight and food intake ( normalized by calorie consumption ) . To investigate changes in the reproductive system , we weighted the testicles of adult and young males . To assess fertility in males , Ubi and Ubi-LepRNull adult males ( n = 5–6/group ) were mated with C57BL6 female mice ( two females for each male ) for 28 days and the number of pregnant females from each group was compared . In females , we monitored the estrous cycle daily for 25 days ( n = 6/group ) . The proportion of cell types in the vaginal smear was determined and the cycle phases were classified as follows: diestrus ( mostly leucocytes ) , metestrus ( mixture of leucocytes and cornified cells ) and proestrus/estrus ( mostly epithelial or cornified cells ) . Commercially available ELISA kits were used to determine the serum concentration of leptin ( Crystal Chem ) , T4 ( Calbiotech ) , testosterone ( Calbiotech ) , insulin ( Crystal Chem ) , and corticosterone ( Arbor Assays ) . Total RNA from the hypothalamus was extracted with TRIzol reagent ( Invitrogen ) . Assessment of RNA quantity and quality was performed with an Epoch Microplate Spectrophotometer ( Biotek ) . Total RNA was incubated with DNase I RNase-free ( Roche Applied Science ) . Reverse transcription was performed with 2 µg of total RNA with SuperScript II Reverse Transcriptase ( Invitrogen ) and random primers p ( dN ) 6 ( Roche Applied Science ) . Real-time polymerase chain reaction was performed using the 7500TM Real-Time PCR System ( Applied Biosystems ) and Power SYBR Green PCR Master Mix ( Applied Biosystems ) . Relative quantification of mRNA was calculated by 2-ΔΔCt . Data were normalized to the geometric average of Actb , Gapdh and Ppia and reported as fold changes compared to values obtained from the control group ( set at 1 . 0 ) . The list of primers is available as Figure 3—source data 2 . The wet brain mass was determined in adult and young mice , both males and females . All behavioral tests were performed in male mice treated with tamoxifen at 10 weeks of age . For the open-field test , mice were placed in an open field arena ( 0 . 3 m ( w ) x 0 . 3 m ( d ) x 0 . 45 m ( h ) for 5 min and monitored by the ANY-maze software , which tracks animal’s position and records the distance travelled . To investigate spatial learning/memory , we performed the Barnes maze test . The maze consists of a white circular platform ( 92 cm of diameter ) elevated 105 cm above the floor with 20 equally spaced holes ( 5 cm diameter and 7 . 5 cm between holes ) along the perimeter with a darkened escape box placed in a constant position under one of the holes at the edge of the platform . Mice are presumed to learn the location of an escape hole using extra-maze visual cues placed on the walls of the room . Mice were initially subjected to one habituation phase in the first day prior to the initial trial . This phase was performed by placing the animal in a cylindrical chamber in the middle of the maze for 10 s . Afterwards , the chamber was removed , and mice were gently guided to the escape box . Animals were allowed to remain in the escape box for 2 min and then were returned to their original cage . The acquisition phase consisted of four trials per day for four days . For each trial , mice were placed in the cylindrical chamber in the middle of the maze for 10 s and then they could explore the maze for 3 min . After trial’s completion ( i . e mice either entered the escape box or remained exploring the maze for 3 min ) animals were gently guided to and remained inside the escape box for 1 min . Subsequently , mice were returned to their home cages until the next trial , with a 15 min interval between trials . Time ( primary latency ) and number of errors ( primary errors ) to reach the escape hole for the first time were recorded by a trained researcher . Errors were considered as the number of head deflections into incorrect holes of the maze and were counted by the experimenter . Test sessions were performed 24 hr and 1 week after the last trial ( days 5 and 12 , respectively ) and consisted of 90 s exploring the maze with the escape box closed . In all experimental steps , the maze was cleaned with 40% ethanol and air-dried before each trial and between mice . The platform was monitored by the ANY-maze software , which recorded the animal’s position and the distance the animal moved ( total distance travelled ) . All results were expressed as mean ±s . e . m . We performed Bartlett’s test of normality to evaluate for Gaussian distribution of samples . When data had normal distribution , we used one-way ANOVA and the Newman-Keuls multiple comparison post-hoc test . In cases of non-parametric data , we used Kruskal-Wallis followed by Dunn’s test . In specific cases , we used the Mann-Whitney test with Bonferroni’s correction . Data was analyzed using two-way ANOVA when appropriate , followed by Newman-Keuls post-hoc test . Statistical analyses were performed using GraphPad Prism software . We considered p values < 0 . 05 to be statistically significant and p<0 . 01 when Bonferroni’s correction was applied .
Leptin regulates energy balance and also exhibits neurotrophic effects during critical developmental periods . However , the actual role of leptin during development is not yet fully understood . To uncover the importance of leptin in early life , the present study restored leptin signaling either at the fourth or tenth week of age in mice formerly null for the leptin receptor ( LepR ) gene . We found that some defects previously considered irreversible due to neonatal deficiency of leptin signaling , including the poor development of arcuate nucleus neural projections , were recovered by LepR reactivation in adulthood . However , LepR deficiency in early life led to irreversible obesity via suppression of energy expenditure . LepR reactivation in adulthood also led to persistent reduction in hypothalamic Pomc , Cartpt and Prlh mRNA expression and to defects in the reproductive system and brain growth . Our findings revealed that early defects in leptin signaling cause permanent metabolic , neuroendocrine and developmental problems .
Leptin is a hormone that keeps us healthy in many ways . It regulates our body weight by reining in our appetite and fine-tuning the energy we burn , and it helps us establish and maintain our fertility . It also participates in brain development . Leptin performs these roles by attaching to specific receptors in nerve cells and relaying relevant information to the brain . Early events can trigger life-long changes in the way our body works , a process called metabolic programming . Leptin is believed to participate in this reprogramming mechanism , but its role remains uncertain . In particular , it is still unclear which leptin-driven changes are permanent , and which ones are reversible . Being able to distinguish between the two types of alterations would help to better grasp the role leptin plays in early development . Here , Ramos-Lobo et al . examined genetically engineered mice born without a working leptin receptor . These animals were impervious to the effects of leptin . Then , once the rodents were adults , they were treated with a drug that restored their leptin receptors , making them sensitive to the hormone again . These experiments revealed that mice without leptin receptors during early life developed obesity , were less able to lose weight and burned less energy . Their reproductive success was also compromised . Finally , the lack of leptin during development caused permanent reduction of the animals’ brains , and changes in the activity of certain genes in the organ . The work by Ramos-Lobo et al . indicates that in mice , lacking leptin sensibility early in life conditions the body to permanently become ‘thrifty’ , burning less energy and making it harder to lose weight . It is rare for humans to be born completely without leptin activity . Yet , having too much or too little food as a baby affects the level of the hormone , or our sensitivity to it: this may permanently change the way our bodies manage energy . Ultimately , learning more about these mechanisms could help us ward off or treat obesity .