Abstract
Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.
Introduction
Proopiomelanocortin (Pomc) and Agouti-related protein (Agrp) expressing neurons in the arcuate nucleus (ARC) of the hypothalamus integrate hormonal signals such as leptin (Cowley et al. 2001), estrogen (Xu et al. 2011) and insulin to control energy and glucose homeostasis (Varela & Horvath 2012). Leptin targets cells expressing multiple leptin receptor isoforms, including a subpopulation of ARC POMC neurons. The full-length leptin receptor (LEPRB) is responsible for intracellular signaling through JAK-STAT signaling pathways (Vaisse et al. 1996, Bates et al. 2003) and mediates leptin-induced satiety responses (Lee et al. 1996, Bates et al. 2003). Although the selective deletion of LepR from POMC neurons in adult mice did not alter food intake, it reduced hepatic glucose production during hyperinsulinemic euglycemic clamps and prevented the fasting-induced fall in leptin levels, independent of changes in fat mass (Caron et al. 2018b).
In addition to LEPRB, there are five truncated forms of leptin receptors generated by alternative splicing of LepR hnRNA. Of these isoforms, LEPRE lacks all intracellular signaling domains (Houseknecht et al. 1996), is produced from the liver and placenta in rodents and is released into the circulation where it binds with high affinity to leptin (Gavrilova et al. 1997). The expression of LepRe in placenta increases as gestation advances (Challier et al. 2003, Zhao et al. 2004). LEPRE might buffer leptin signaling by reducing its renal clearance under normal physiological conditions and prevent excessive binding of elevated circulating leptin to LEPRB in extreme metabolic conditions, such as late pregnancy (Tu et al. 2008, Trujillo et al. 2011). Leptin receptors in the placenta might also influence fetal growth by mediating leptin transport to the fetal circulation or by local leptin bioactivity (Smith & Waddell 2002).
Pregnancy and lactation represent allostatic states in which the neuronal circuits controlling feeding behavior adapt to the high demands for nutrients from the developing fetuses and suckling pups. In particular, lactation is associated with marked hyperphagia, increased expression of orexigenic neuropeptides in the hypothalamus, transient hyperleptinemia and reduced transport of leptin into the brain (Gustafson et al. 2019). The maternal adaptations to pregnancy are mediated largely by the placenta, which separates the maternal and fetal circulations and secretes hormones including placental lactogens, progesterone and estrogen to maintain pregnancy (Watson & Cross 2005, Ladyman et al. 2010, Napso et al. 2018). Similar to LepR, Pomc is expressed in both the placenta and hypothalamic neurons (Chen et al. 1986, Challier et al. 2003). The human POMC transcript is localized to trophoblast cells and its expression can be detected from the first to the final trimester with increased levels before parturition (Cooper et al. 1996). However, the function and regulation of POMC expression in the placenta is poorly understood.
Previous work from our lab identified two phylogenetically conserved neuronal Pomc enhancers named nPE1 and nPE2, located approximately 12 kb and 10 kb upstream of the mouse Pomc promoter (de Souza et al. 2005, Franchini et al. 2011, Lam et al. 2015). Both enhancers are required for maximal transcription of Pomc in the ARC, but not pituitary corticotrophs or melanotrophs. Individual deletions of nPE1 or nPE2 reduced hypothalamic Pomc expression by ~70% or ~20%, respectively (Lam et al. 2015). nPE2 is an ancient Pomc enhancer that originated more than 166 million years ago in the lineage leading to all extant mammals (Santangelo et al. 2007), whereas nPE1 is present only in the genomes of placental mammals and originated at least 24 million years later from the exaptation of a distinct retroposon (Franchini et al. 2011). Compared to non-placental animals, for example, Monotremes and Marsupials, the existence of a complex placenta allows the nourishment of young during a longer gestational period and thus the birth of more mature and well developed young (Ferner et al. 2017). Although Pomc is highly expressed in the placenta (Zhu & Pintar 1998), it is unknown whether the recent evolutionary association of nPE1 to the Pomc gene in placental mammals is coincidental or indicative of a regulatory role for placental Pomc transcription.
Because of the importance of leptin and POMC peptides in the central regulation of energy balance in adult animals and the parallels between hypothalamic and placental LepR and Pomc expression, we hypothesized that deletion of nPE1 from the distal enhancer locus of Pomc would alter expression of Pomc in the placenta and disrupt the normal metabolic adaptations to pregnancy and lactation. Moreover, previous studies of pregnant rats (Khorram et al. 1984, Mann et al. 1997, Ladyman et al. 2009, Trujillo et al. 2011) showed reduced hypothalamic expression of Pomc in late pregnancy and early postnatal days, possibly contributing to hyperphagia by reducing satiety. Therefore, we questioned whether constitutive reductions of Pomc expression in the ARC of Pomc∆1/∆1 dams would further exacerbate the hyperphagia and weight gain during pregnancy and lactation.
Materials and methods
Animal care
All experiments were approved by the University of Michigan’s Institutional Animal Care and Use Committee and followed the Public Health Service guidelines for the humane care and use of experimental animals. Mice were housed in ventilated cages under controlled temperature and photoperiod (12 h light:12 h darkness cycle), with tap water and laboratory chow (5L0D, LabDiet, St. Louis, MO, USA) containing 28.67% protein, 13.38% fat and 57.94% carbohydrate available ad libitum. Breeding mice were fed with a breeder chow diet (5008, LabDiet) containing 26.53% protein, 16.97% fat and 56.50% carbohydrates.
Generation and breeding of mice
Mice lacking nPE1 (Pomc∆1/∆1) were generated by homologous recombination in embryonic stem cells and backcrossed for 12–14 generations onto the C57BL/6J genetic background as described previously (Lam et al. 2015). In the present study, adult (9–20 wk old) virgin female Pomc+/+and Pomc∆1/∆1 mice were bred with Pomc+/+ male mice and the day of copulation plug detection was counted as day post coitum (dpc) 0.5. The genotypes of offspring from these matings were either Pomc+/+ or Pomc∆1/+, respectively. A separate cohort of virgin Pomc∆1/∆1 females was bred with Pomc∆1/∆1 males to generate homozygous Pomc∆1/∆1 offspring, and these Pomc∆1/∆1 dams were denoted as Pomcho∆1/∆1to indicate that all their pups were also homozygous for the hypomorphic allele. Pregnant dams were housed individually and their body weights were recorded on the mornings of dpc3.5, dpc6.5, dpc9.5, dpc12.5 and dpc18.5 with continued measurements at postpartum days (ppd) 0, 3, 6, 9, 12, 15, 18 and 21. Twenty-four-hour food intake was assessed during early pregnancy (dpc3.5–dpc4.5), late pregnancy (dpc17.5–dpc18.5) and mid-lactation (ppd10–ppd11). Body composition of the dams was assessed by NMR (Minispec, Bruker Instruments) before pregnancy, at late pregnancy (dpc17.5 and dpc18.5) and at days ppd0 and ppd21. The number of pups per litter was recorded at birth, and the body weight of each pup was measured at ppd0, ppd10 and ppd21.
Plasma leptin and soluble leptin receptor measurements
Blood was collected into EDTA tubes from virgin females and dams at dpc17.5 or dpc18.5, ppd0 and ppd21. Plasma leptin levels were determined by ELISA (MOB00, Mouse/Rat Leptin Quantikine ELISA Kit, R&D Systems) according to the manufacturer’s instructions, which included acidification/urea/neutralization steps of the dpc17.5, dpc18.5 and ppd0 samples to fully recover leptin from LEPRE interference by denaturation of the binding protein. Soluble leptin receptor (LEPRE) was measured by ELISA (DY497, Mouse Leptin R DuoSet ELISA, R&D Systems) according to the manufacturer’s instructions.
Histology
Placentas were dissected at dpc18.5 and post-fixed in 10% neutral buffered formalin overnight at 4°C. Samples were then washed three times in 1× phosphate buffered saline and transferred to 70% ethanol for short-term storage. Paraffin-embedding, sample sectioning (5 µm) and hematoxylin and eosin (H&E) staining were performed by the University of Michigan Comprehensive Cancer Center Research Histology and Immunohistochemistry Core. Images were acquired using Nikon Eclipse 90i microscope equipped with NIS-Elements AR software.
Quantitative RT-PCR
Total RNA was prepared from arcuate nucleus and placenta with TRIzol (15596026, ThermoFisher Scientific) as described by the manufacturer. cDNA was obtained by RT of 1 µg RNA with random primers using M-MLV Reverse Transcriptase (28025013, ThermoFisher Scientific). qRT-PCR was performed on all samples using a StepOne Real Time PCR System (Applied Biosystems) and SYBR Green Master Mix (4385612, ThermoFisher Scientific). The primers for detection of Pomc, Agrp, pan leptin receptor (LepR), LepRa, LepRb, LepRe, Plac1, Tpbpa and reference genes Hprt and Tbp are in Supplementary Table 1 (see section on supplementary materials given at the end of this article). All primers were used at a final concentration of 250 nM. The results were analyzed using the 2−ΔΔCT relative quantitation method and graphed as a percentage of the average data from the WT control females.
Statistical analysis
Two-tailed Student’s t-test or the Mann–Whitney t-test for nonparametric data were used to determine the significant differences between pairs of genotypes for one dependent variable. Two-way ANOVAs followed by Bonferroni’s post-hoc multiple comparisons tests for pair-wise comparisons were performed to determine the effects of genotype and developmental stages. Linear correlation and regression analyses were performed to determine the relationship between plasma leptin and body fat mass at different physiological stages. Normalized placental weights, placental diameters, fetal weights and crown-rump lengths were calculated by averaging the reading from three measured placentas and fetuses in each litter, divided by the litter size and multiplied by the average litter size for each genotype (Pomc+/+: 7.36 and Pomc∆1/∆1 : 7.27). A detailed description of the statistical method used for each analysis is noted in each figure legend. P < 0.05 was considered significant.
Results
Maternal deletion of nPE1 from Pomc did not affect the normal temporal patterns or magnitude of changes in body weight or food intake during gestation and lactation
The weight curves comparing Pomc∆1/∆1 to Pomc+/+ dams (Fig. 1A) were indistinguishable from each other. Pregnancy requires increased food intake to meet the energy requirements for offspring to develop and grow (Makarova et al. 2010). Compared to dpc3.5, the daily food intake at dpc17.5 increased slightly in both genotypes, but without reaching statistical significance (Pomc+/+: 4.1 ± 0.1 to 4.8 ± 0.2 g; and Pomc∆1/∆1: 4.5 ± 3.0 to 4.7 ± 0.3 g) (Fig. 1B). However, lactation dramatically increased the dams’ daily food intake, measured from ppd10 to ppd11 (F2,81 = 92.1, P < 0.0001; two-way ANOVA, main effect of age), but again, there were no significant genotype differences (Pomc+/+: 11.1 ± 0.5 g; Pomc∆1/∆1: 10.1 ± 0.9 g) or genotype by age interactions (Fig. 1B).
Targeted deletion of neuronal Pomc enhancer nPE1 reduced total body fat mass during pregnancy but not body weight during pregnancy or lactation. (A) Comparison of body weight changes across gestation (n = 16–17) and lactation (n = 6–8) between Pomc+/+ and Pomc∆1/∆1 dams. (B) Comparisons of 24 hr food intake at early pregnancy (dpc3.5–dpc4.5, n = 17–18), late pregnancy (dpc17.5–dpc18.5, n = 14–15) and mid lactation (ppd10–ppd11, n = 11–12). Body composition comparisons between Pomc+/+ and Pomc∆1/∆1 dams (C and D) at the indicated stages (n = 10–17). Two-way ANOVAs followed by Bonferroni’s post-hoc multiple comparison tests for pair-wise comparisons of food intake, fat mass and lean mass (A, B, C and D). Pomc∆1/∆1 dams exhibited a significant reduction in fat mass during gestation from NP to ppd0 (E) while control Pomc+/+ dams had no change in fat mass during gestation (F). Paired Student’s two-tailed t-tests were used to determine the significant genotype differences in fat mass from NP to ppd0 (E and F). NP, non-pregnant; dpc, days post coitum; ppd, postpartum day. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Targeted deletion of neuronal Pomc enhancer nPE1 reduced total body fat mass during pregnancy but not body weight during pregnancy or lactation. (A) Comparison of body weight changes across gestation (n = 16–17) and lactation (n = 6–8) between Pomc+/+ and Pomc∆1/∆1 dams. (B) Comparisons of 24 hr food intake at early pregnancy (dpc3.5–dpc4.5, n = 17–18), late pregnancy (dpc17.5–dpc18.5, n = 14–15) and mid lactation (ppd10–ppd11, n = 11–12). Body composition comparisons between Pomc+/+ and Pomc∆1/∆1 dams (C and D) at the indicated stages (n = 10–17). Two-way ANOVAs followed by Bonferroni’s post-hoc multiple comparison tests for pair-wise comparisons of food intake, fat mass and lean mass (A, B, C and D). Pomc∆1/∆1 dams exhibited a significant reduction in fat mass during gestation from NP to ppd0 (E) while control Pomc+/+ dams had no change in fat mass during gestation (F). Paired Student’s two-tailed t-tests were used to determine the significant genotype differences in fat mass from NP to ppd0 (E and F). NP, non-pregnant; dpc, days post coitum; ppd, postpartum day. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Targeted deletion of neuronal Pomc enhancer nPE1 reduced total body fat mass during pregnancy but not body weight during pregnancy or lactation. (A) Comparison of body weight changes across gestation (n = 16–17) and lactation (n = 6–8) between Pomc+/+ and Pomc∆1/∆1 dams. (B) Comparisons of 24 hr food intake at early pregnancy (dpc3.5–dpc4.5, n = 17–18), late pregnancy (dpc17.5–dpc18.5, n = 14–15) and mid lactation (ppd10–ppd11, n = 11–12). Body composition comparisons between Pomc+/+ and Pomc∆1/∆1 dams (C and D) at the indicated stages (n = 10–17). Two-way ANOVAs followed by Bonferroni’s post-hoc multiple comparison tests for pair-wise comparisons of food intake, fat mass and lean mass (A, B, C and D). Pomc∆1/∆1 dams exhibited a significant reduction in fat mass during gestation from NP to ppd0 (E) while control Pomc+/+ dams had no change in fat mass during gestation (F). Paired Student’s two-tailed t-tests were used to determine the significant genotype differences in fat mass from NP to ppd0 (E and F). NP, non-pregnant; dpc, days post coitum; ppd, postpartum day. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Deletion of nPE1 from Pomc significantly reduced maternal body fat mass during gestation
We measured body composition of the females before pregnancy (NP), on the day of parturition (ppd0) and on the day of weaning (ppd21). A two-way ANOVA for lean mass revealed a significant main effect across the three time points (F2,83 = 80.3, P < 0.0001), but not for genotype or time by genotype interactions (Fig. 1C). A significant main effect across time was also detected for fat mass (Pomc +/+vs. Pomc∆1/∆1: F2,81 = 3.5, P = 0.03) (Fig. 1D). There was an additional significant main effect of genotype for fat mass (F1,81 = 8.8, P = 0.004) but no time by genotype interaction. Follow-up post-hoc multiple comparisons tests indicated that non-pregnant Pomc∆1/∆1females had 31% greater fat mass (Pomc∆1/∆1: 4.15 ± 0.23 g; Pomc+/+: 3.17 ± 0.22 g; P = 0.018) compared to their sibling Pomc+/+non-pregnant controls as reported previously (Lam et al. 2015) (Fig. 1D). Unexpectedly, unlike Pomc+/+ dams that had no change in their fat mass during gestation, the Pomc∆1/∆1dams had significantly reduced fat mass by parturition. The preservation of fat mass for Pomc+/+ dams during pregnancy, but reduction in fat mass for the Pomc∆1/∆1dams, are shown alternatively with the repeated measures connected by lines for the individual mice in Fig. 1E and F, respectively. Body composition measurements from an independent cohort of mice at dpc17.5 and dpc18.5 also showed a significant increase of lean mass (Supplementary Fig. 1A) in both genotypes but confirmed a remarkable reduction of fat mass (Supplementary Fig. 1B, C and D) only in Pomc∆1/∆1 dams during gestation. Dams of both genotypes had similar significant increases in fat mass during lactation (ppd0 to ppd21) (Fig. 1D).
Deletion of nPE1 from Pomc did not impair placental growth
Because nPE1 is present exclusively in the genome of placental mammals, we tested its physiological significance by evaluating placental weights, diameters, morphology and function at 18.5 days of pregnancy, a time when the placenta is structurally fully developed and at its maximum weight. Deletion of nPE1 from Pomc did not change placental weights or diameters (Fig. 2A and B). Vertical hemisections of the placenta revealed three layers: the labyrinth zone (Lab), the junctional zone (JunZ) and maternal decidua basalis (Dec). Deletion of nPE1 from Pomc did not change the integrity of these three components in the mature placenta (Fig. 2C). To further assess the requirements of the nPE1 Pomc enhancer on the function of placenta, we measured the expression of Pomc and two essential genes for placental growth and development. Relative quantification by RT-PCR indicated that deletion of nPE1 did not change Pomc mRNA levels in placenta, indicating that nPE1 is not required for placental Pomc expression, unlike in the ARC, but similar to pituitary (Fig. 2D). Placenta-specific 1 (Plac1) expression is almost entirely restricted to trophoblast cells (Cocchia et al. 2000), and its deficiency leads to placentomegaly and intrauterine growth retardation (Jackman et al. 2012). Likewise, ablation of Trophoblast-specific protein alpha (Tpbpa) positive cells reduced maternal spiral artery sizes and decreased blood flow in the labyrinth and further induced embryonic death after dpc10.5 (Hu & Cross 2011). However, expression levels of Plac1 and Tpbpa in the placenta did not differ by genotype as determined by quantitative RT-PCR (Fig. 2E and F).
Pregnant mice with neuronal Pomc enhancer nPE1 deletion had normal placental morphology and function. Three placentas from each dam were measured and the average weight (A) and diameter (B) were compared between Pomc+/+ and Pomc∆1/∆1 (n = 8–12 dams). Placental morphology was characterized by Hematoxylin and Eosin (H&E) staining (C). Dec, Decidua; JunZ, Junctional Zone; Lab, Labyrinth. Quantitative RT-PCR was performed to compare Pomc (D), Plac1 (Placenta specific 1) (E) and Tpbpa (Trophoblast specific protein alpha) (F) gene expression in placenta (n = 8–12 dams). Unpaired Student’s two-tailed t-tests revealed no significant genotype differences for any of the variables. Data shown are the mean ± s.e.m.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Pregnant mice with neuronal Pomc enhancer nPE1 deletion had normal placental morphology and function. Three placentas from each dam were measured and the average weight (A) and diameter (B) were compared between Pomc+/+ and Pomc∆1/∆1 (n = 8–12 dams). Placental morphology was characterized by Hematoxylin and Eosin (H&E) staining (C). Dec, Decidua; JunZ, Junctional Zone; Lab, Labyrinth. Quantitative RT-PCR was performed to compare Pomc (D), Plac1 (Placenta specific 1) (E) and Tpbpa (Trophoblast specific protein alpha) (F) gene expression in placenta (n = 8–12 dams). Unpaired Student’s two-tailed t-tests revealed no significant genotype differences for any of the variables. Data shown are the mean ± s.e.m.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Pregnant mice with neuronal Pomc enhancer nPE1 deletion had normal placental morphology and function. Three placentas from each dam were measured and the average weight (A) and diameter (B) were compared between Pomc+/+ and Pomc∆1/∆1 (n = 8–12 dams). Placental morphology was characterized by Hematoxylin and Eosin (H&E) staining (C). Dec, Decidua; JunZ, Junctional Zone; Lab, Labyrinth. Quantitative RT-PCR was performed to compare Pomc (D), Plac1 (Placenta specific 1) (E) and Tpbpa (Trophoblast specific protein alpha) (F) gene expression in placenta (n = 8–12 dams). Unpaired Student’s two-tailed t-tests revealed no significant genotype differences for any of the variables. Data shown are the mean ± s.e.m.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Constitutive deletion of nPE1 from Pomc did not induce any growth alterations in offspring
We next characterized the growth and development of offspring from Pomc∆1/∆1 and Pomc+/+ dams. Compared to the Pomc+/+pups from Pomc+/+ dams, the Pomc∆1/+ pups from Pomc∆1/∆1 dams had indistinguishable numbers per litter at birth and per sex at weaning (Fig. 3A and B), the same body weights from birth (ppd0) until weaning at ppd21 (Fig. 3C) and similar crown-rump lengths at birth (Pomc+/+: 21.4 ± 0.2; Pomc∆1/∆1: 20.9 ± 0.3 mm) (Fig. 3D).
Maternal deletion of neuronal Pomc enhancer nPE1 did not cause any growth defects in the offspring. (A) Average number of pups per litter (n = 17–19 litters), (B) average number of male/female pups per litter (n = 10–16 litters), (C) body weight of pups (n = 10–17 litters) at the indicated postnatal days (ppd) and (D) Crown-Rump lengths from dpc18.5 fetuses (n = 11–12 litters) were compared between genotypes. Unpaired two-tailed Student’s t-tests revealed no significant genotype differences for any of the variables. Data shown are the mean ± s.e.m.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Maternal deletion of neuronal Pomc enhancer nPE1 did not cause any growth defects in the offspring. (A) Average number of pups per litter (n = 17–19 litters), (B) average number of male/female pups per litter (n = 10–16 litters), (C) body weight of pups (n = 10–17 litters) at the indicated postnatal days (ppd) and (D) Crown-Rump lengths from dpc18.5 fetuses (n = 11–12 litters) were compared between genotypes. Unpaired two-tailed Student’s t-tests revealed no significant genotype differences for any of the variables. Data shown are the mean ± s.e.m.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Maternal deletion of neuronal Pomc enhancer nPE1 did not cause any growth defects in the offspring. (A) Average number of pups per litter (n = 17–19 litters), (B) average number of male/female pups per litter (n = 10–16 litters), (C) body weight of pups (n = 10–17 litters) at the indicated postnatal days (ppd) and (D) Crown-Rump lengths from dpc18.5 fetuses (n = 11–12 litters) were compared between genotypes. Unpaired two-tailed Student’s t-tests revealed no significant genotype differences for any of the variables. Data shown are the mean ± s.e.m.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Pomc, Agrp and LepR gene expression in the arcuate nucleus
In order to understand possible mechanisms underlying hyperphagia during late pregnancy and lactation, we measured expression of relevant genes in the arcuate nucleus, a crucial site in the hypothalamus for regulating appetite and body weight. A two-way ANOVA indicated a significant main effect of maternal genotype for Pomc expression (F1,73 = 65.57, P < 0.0001) (Fig. 4A). Specifically, Pomc∆1/∆1 dams exhibited an average of 71% reduction in Pomc expression, relative to Pomc+/+ dams collapsed across all of the interrogated developmental stages, which is consistent with our previous report based on virgin mice (Lam et al. 2015). Agrp expression was increased ~2.4-fold at dpc18.5 and ~2.9-fold at ppd10 and returned to normal levels at ppd21 in both genotypes compared to their non-pregnant controls (Two-way ANOVA, main effect of physiological stage: F3,73 = 23.28, P < 0.0001) (Fig. 4B). There was a significant increase in pan-LepR expression in both genotypes at dpc18.5 compared to both non-pregnant mice and lactating dams at ppd10 (Fig. 4C). There were no main effects of genotype, stage or interactions between the two independent variables for either LepRa or LepRb (Fig. 4D and E).
Gene expression in the hypothalamic arcuate nucleus measured by qRT-PCR from Pomc∆1/∆1 mice relative to Pomc+/+ controls before pregnancy, at late gestation, at lactation and at weaning. Comparison of relative gene expression for Pomc (A), Agrp (B) and leptin receptor isoforms (C, D and E) from the two genotypes at indicated stages (n = 8–17 dams). NP, not pregnant; dpc, day post coitum; ppd, postpartum day. Two-way ANOVAs followed by Bonferroni’s post-hoc multiple pair-wise comparison tests were performed. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Gene expression in the hypothalamic arcuate nucleus measured by qRT-PCR from Pomc∆1/∆1 mice relative to Pomc+/+ controls before pregnancy, at late gestation, at lactation and at weaning. Comparison of relative gene expression for Pomc (A), Agrp (B) and leptin receptor isoforms (C, D and E) from the two genotypes at indicated stages (n = 8–17 dams). NP, not pregnant; dpc, day post coitum; ppd, postpartum day. Two-way ANOVAs followed by Bonferroni’s post-hoc multiple pair-wise comparison tests were performed. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Gene expression in the hypothalamic arcuate nucleus measured by qRT-PCR from Pomc∆1/∆1 mice relative to Pomc+/+ controls before pregnancy, at late gestation, at lactation and at weaning. Comparison of relative gene expression for Pomc (A), Agrp (B) and leptin receptor isoforms (C, D and E) from the two genotypes at indicated stages (n = 8–17 dams). NP, not pregnant; dpc, day post coitum; ppd, postpartum day. Two-way ANOVAs followed by Bonferroni’s post-hoc multiple pair-wise comparison tests were performed. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Deletion of nPE1 from Pomc significantly increased maternal plasma leptin levels at dpc18.5 independent of fat mass
Pregnancy is associated with increased levels of maternal leptin in women and rats, but the increases are particularly marked in mice (Masuzaki et al. 1997, Malik et al. 2005). Moreover, central Pomc-expressing neurons directly regulate peripheral leptin release through activation of sympathetic efferents projecting to white adipocytes (Rayner & Trayhurn 2001, Caron et al. 2018b ). Therefore, we measured circulating leptin in control Pomc+/+ and Pomc∆1/∆1dams and conducted correlation analyses between circulating leptin and fat mass before pregnancy and at dpc18.5 to elucidate possible roles of the central hypomorphic Pomc allele on the modulation of circulating leptin levels. Circulating leptin was substantially increased at dpc18.5 in Pomc+/+ dams compared to non-pregnant females, remained elevated at ppd0 and dropped to below non-pregnant levels by ppd10 (Fig. 5A). Pomc∆1/∆1 females exhibited a similar temporal pattern for circulating leptin, but the levels were significantly higher in Pomc∆1/∆1 mice before pregnancy and at dpc18.5 by ~2-fold compared to the control dams (Pomc+/+vs. Pomc∆1/∆1: NP, 4.6 ± 0.4 vs 10.9 ± 1.5; dpc18.5, 75.8 ± 9.0 vs 147.8 ± 25.5; ppd0, 72.5 ± 12.2 vs 97.9 ± 29.5; ppd10, 3.1 ± 0.4 vs 6.0 ± 1.0 ng/mL). Despite the quantitative differences, linear regression analyses showed that leptin levels were significantly correlated with total fat mass in both Pomc+/+ and Pomc∆1/∆1 females before pregnancy (Pomc+/+: R2 = 0.468, P = 0.0291 vs. Pomc∆1/∆1: R2 = 0.881, P = 0.0017) (Fig. 5B). The similar regression line slopes and Y intercepts between the two genotypes before pregnancy indicate a consistent proportionality between total fat mass and circulating leptin levels for Pomc+/+ and Pomc∆1/∆1 females. However, the slopes of the regression lines were significantly more shallow at dpc18.5 compared to virgin mice for both genotypes (Pomc+/+: NP, 0.2221 ± 0.08 vs dpc18.5, 0.0036 ± 0.005, P = 0.0512; Pomc∆1/∆1: NP, 0.2419 ± 0.04 vs dpc18.5, 0.0051 ± 0.004, P = 0.0010) even though total fat mass was not changed between these time points in the WT controls (NP, 3.24 ± 0.12 vs dpc18.5, 3.07 ± 0.13 g) and was actually decreased in the Pomc∆1/∆1 dams (NP, 4.83 ± 0.38 vs dpc18.5, 3.90 ± 0.24 g) (Supplementary Fig. 1B).
Deletion of neuronal Pomc enhancer nPE1 significantly increased plasma leptin levels at dpc18.5 independent of total body fat mass. (A) Serial plasma leptin levels at indicated experimental stages (n = 7–10 dams). Two-way ANOVAs followed by Bonferroni’s post-hoc multiple pair-wise comparison tests and unpaired two-tailed Student’s t-tests were performed to evaluate the genotype effect. Data shown are the mean ± s.e.m. **P < 0.01 and ***P < 0.005. (B) Correlation and linear regression analyses of plasma leptin with respect to body fat mass in non-pregnant (NP) and dpc18.5 mice (n = 6–10 dams). The P-values indicate whether regression line slopes are significantly different from 0.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Deletion of neuronal Pomc enhancer nPE1 significantly increased plasma leptin levels at dpc18.5 independent of total body fat mass. (A) Serial plasma leptin levels at indicated experimental stages (n = 7–10 dams). Two-way ANOVAs followed by Bonferroni’s post-hoc multiple pair-wise comparison tests and unpaired two-tailed Student’s t-tests were performed to evaluate the genotype effect. Data shown are the mean ± s.e.m. **P < 0.01 and ***P < 0.005. (B) Correlation and linear regression analyses of plasma leptin with respect to body fat mass in non-pregnant (NP) and dpc18.5 mice (n = 6–10 dams). The P-values indicate whether regression line slopes are significantly different from 0.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Deletion of neuronal Pomc enhancer nPE1 significantly increased plasma leptin levels at dpc18.5 independent of total body fat mass. (A) Serial plasma leptin levels at indicated experimental stages (n = 7–10 dams). Two-way ANOVAs followed by Bonferroni’s post-hoc multiple pair-wise comparison tests and unpaired two-tailed Student’s t-tests were performed to evaluate the genotype effect. Data shown are the mean ± s.e.m. **P < 0.01 and ***P < 0.005. (B) Correlation and linear regression analyses of plasma leptin with respect to body fat mass in non-pregnant (NP) and dpc18.5 mice (n = 6–10 dams). The P-values indicate whether regression line slopes are significantly different from 0.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Increased placental LepRe but decreased pan-LepR and LepRb mRNA in Pomc∆1/∆1 dams
In humans and rats, the placenta has been recognized, in addition to adipose tissue, as one of the sources for circulating leptin (Masuzaki et al. 1997, Amico et al. 1998). In contrast, mouse placenta does not produce or release leptin as shown by several previous studies (Tomimatsu et al. 1997, Zhao et al. 2003). We also failed to detect any leptin transcripts in placenta at dpc18.5 (data not shown), indicating that the placenta is not a direct source of leptin secretion and therefore not responsible for the elevated leptin in pregnant mice. Leptin receptors, on the other hand, are expressed in the placenta across different species (Smith & Waddell 2002, Challier et al. 2003, Zhao et al. 2004). In particular, the soluble form of leptin receptor (LEPRE) is produced by the placenta and released into the circulation during pregnancy (Lammert et al. 2002). Pomc ∆1/∆1 mice exhibited significantly reduced transcript levels of placental pan LepR and LepRb compared to Pomc+/+dams, but LepRa mRNA levels were not different between the genotypes (Fig. 6). However, the transcript abundance of LepRe in placentas from Pomc∆1/∆1 dams was ~30% greater than that from Pomc+/+ dams and approached statistical significance (P = 0.057) (Fig. 6).
Inactivation of Pomc enhancer nPE1 increased soluble leptin receptor (LepRe) expression in the placenta at late pregnancy stage (dpc18.5). Quantitative RT-PCR was performed to detect expression of leptin receptor isoforms in placenta at dpc18.5 (n = 8–12 dams). Two-tailed Student’s t-tests were performed to determine any significant differences between genotypes. Data shown are the mean ± s.e.m. ***P < 0.005 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Inactivation of Pomc enhancer nPE1 increased soluble leptin receptor (LepRe) expression in the placenta at late pregnancy stage (dpc18.5). Quantitative RT-PCR was performed to detect expression of leptin receptor isoforms in placenta at dpc18.5 (n = 8–12 dams). Two-tailed Student’s t-tests were performed to determine any significant differences between genotypes. Data shown are the mean ± s.e.m. ***P < 0.005 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Inactivation of Pomc enhancer nPE1 increased soluble leptin receptor (LepRe) expression in the placenta at late pregnancy stage (dpc18.5). Quantitative RT-PCR was performed to detect expression of leptin receptor isoforms in placenta at dpc18.5 (n = 8–12 dams). Two-tailed Student’s t-tests were performed to determine any significant differences between genotypes. Data shown are the mean ± s.e.m. ***P < 0.005 and ****P < 0.0001.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
To investigate whether the elevated transcript levels of LepRe led to increased circulating soluble leptin receptor, we measured plasma leptin receptor levels using an ELISA kit which was validated in a previous in vitro study involving cell lines transfected with various tagged leptin receptor constructs (Gan et al. 2012). Compared to non-pregnant mice, dams at dpc18.5 exhibited a highly significant 2.2-fold increase in soluble leptin receptor in both genotypes (ordinary two-way ANOVA: F(1,30) = 18.03, P = 0.0002, main effect of stage), which is consistent with the previous findings that placenta releases soluble leptin receptor during pregnancy (Yamaguchi et al. 1998). However, deletion of nPE1 did not induce higher circulating LEPRE compared to WT control mice (Supplementary Fig. 2). These data revealed the lack of any obvious linear stoichiometry for circulating leptin and LepRe levels between nonpregnant and dpc18.5 dams of either genotype.
Breeding of Pomc∆1/∆1 females with homozygous Pomc∆1/∆1 males increased circulating leptin and LepRe mRNA abundance in placenta at dpc18.5
The placenta is composed of fetal-derived components, such as trophoblast cells, and maternal uterine tissue that undergoes a specialized decidualization reaction to support normal placentation (Rossant & Cross 2001, Woods et al. 2018). Because our breeding strategy (♀ Pomc∆1/∆1 x ♂ Pomc+/+) yielded all Pomc∆1/+ pups, we set up an additional breeding scheme in which female Pomc∆1/∆1were mated with male Pomc∆1/∆1 mice to detect any possible effects from the Pomc∆1/∆1 fetuses on placental maturation and the maternal circulation. Compared to the previously described Pomc+/+and Pomc∆1/∆1 dams (Fig. 2A and C), Pomcho∆1/∆1 dams had similar placental size and placental weights (Fig. 7A). The combination of Pomc∆1/∆1 dams and fetuses did not change placental integrity as indicated by H&E staining (Fig. 7B). After normalization to litter size, the fetal lengths and fetal weights were similar between the two genotypes (Fig. 7C). The two breeding schemes generated similar numbers of pups per litter (Fig. 7D). qRT-PCR analysis of gene expression from the Pomcho∆1/∆1 placentas showed significant reductions in mRNA levels for both pan LepR and LepRa mRNA compared to Pomc+/+ dams. LepRb mRNA levels were not changed, but LepRe levels were increased by 30% in the placentas of Pomcho∆1/∆1 compared to Pomc+/+ dams (Fig. 7E), similar to the results shown in Fig. 6 for Pomc∆1/∆1 dams. There were no genotype differences in placental expression of Pomc, Tpbpa or Plac1. The Pomcho∆1/∆1 dams exhibited a significant increase in circulating leptin at dpc18.5 relative to Pomc +/+dams (Pomcho∆1/∆1: 103.0 ± 12.5 ng/mL; Pomc+/+: 44.2 ± 4.1; Mann Whitney two-tailed test; U = 9, P = 0.0003) (Fig. 7F), similar to the Pomc∆1/∆1 dams described previously in this report (Fig. 5A). Blood samples from an independent cohort of Pomcho∆1/∆1dams between the broader stage range of dpc16.5 to ppd0 confirmed that leptin levels at late pregnancy to parturition were significantly higher in the Pomcho∆1/∆1 compared to Pomc+/+ dams (Pomc+/+ : 28.3 ± 3.5; Pomcho∆1/∆1: 72.0 ± 14.8 ng/mL; Mann–Whitney two-tailed test; U = 16, P = 0.0032).
Pomcho∆1/∆1 dams had increased plasma leptin levels and LepRe expression in placenta. Comparison of normalized placental diameters, normalized placental weights (A) and placental morphology (B) (n = 11 dams) between Pomc+/+ and Pomcho∆1/∆1 dams. Three placentas from each dam were measured. The normalized value was calculated by averaging three measurements, dividing by the number of pups per litter and multiplying by the average number of pups per litter from each genotype. (C) Normalized Crown-Rump lengths, normalized fetal weights and (D) number of pups per litter (n = 11 litter) at dpc18.5 from Pomc+/+ and Pomcho∆1/∆1 dams. (E) Gene expression profiles in Pomcho∆1/∆1 placentas (n = 10–11 dams). (F) Comparison of plasma leptin levels between Pomc+/+ and Pomcho∆1/∆1 (n = 10–11 dams) at dpc18.5. Data outliers were identified and removed using the ROUT method in Prism8. Each data set was analyzed by unpaired two-tail t-tests to identify genotype differences. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.005.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Pomcho∆1/∆1 dams had increased plasma leptin levels and LepRe expression in placenta. Comparison of normalized placental diameters, normalized placental weights (A) and placental morphology (B) (n = 11 dams) between Pomc+/+ and Pomcho∆1/∆1 dams. Three placentas from each dam were measured. The normalized value was calculated by averaging three measurements, dividing by the number of pups per litter and multiplying by the average number of pups per litter from each genotype. (C) Normalized Crown-Rump lengths, normalized fetal weights and (D) number of pups per litter (n = 11 litter) at dpc18.5 from Pomc+/+ and Pomcho∆1/∆1 dams. (E) Gene expression profiles in Pomcho∆1/∆1 placentas (n = 10–11 dams). (F) Comparison of plasma leptin levels between Pomc+/+ and Pomcho∆1/∆1 (n = 10–11 dams) at dpc18.5. Data outliers were identified and removed using the ROUT method in Prism8. Each data set was analyzed by unpaired two-tail t-tests to identify genotype differences. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.005.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Pomcho∆1/∆1 dams had increased plasma leptin levels and LepRe expression in placenta. Comparison of normalized placental diameters, normalized placental weights (A) and placental morphology (B) (n = 11 dams) between Pomc+/+ and Pomcho∆1/∆1 dams. Three placentas from each dam were measured. The normalized value was calculated by averaging three measurements, dividing by the number of pups per litter and multiplying by the average number of pups per litter from each genotype. (C) Normalized Crown-Rump lengths, normalized fetal weights and (D) number of pups per litter (n = 11 litter) at dpc18.5 from Pomc+/+ and Pomcho∆1/∆1 dams. (E) Gene expression profiles in Pomcho∆1/∆1 placentas (n = 10–11 dams). (F) Comparison of plasma leptin levels between Pomc+/+ and Pomcho∆1/∆1 (n = 10–11 dams) at dpc18.5. Data outliers were identified and removed using the ROUT method in Prism8. Each data set was analyzed by unpaired two-tail t-tests to identify genotype differences. Data shown are the mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.005.
Citation: Journal of Endocrinology 245, 1; 10.1530/JOE-19-0576
Discussion
In this study, we investigated the effects of a hypomorphic Pomc allele created by a germ-line deletion of the neuronal-specific Pomc enhancer nPE1 on the regulation of maternal metabolism, expression of Pomc in the placenta, circulating leptin in the dams, placental growth during pregnancy, litter size and pup growth during lactation. nPE1 originated from an exaptation event of a mammalian-apparent LTR retrotransposon sometime between the metatherian/eutherian split (147 Mya) and the placental mammalian radiation (~90 Mya). Therefore, nPE1 is a placental apomorphy that is located exclusively in the genomes of all placental mammals, but not other vertebrates, including the non-placental mammalian orders Monotremata and Marsupialia (Franchini et al. 2011). The other known Pomc enhancer nPE2 appeared earlier in evolution from an independent exaptation event from a different retrotransposon and is found in all mammals (Santangelo et al. 2007). nPE1 has one bona fide tandem binding motif for the transcription factors ISLET1 and NKX2.1, and both factors are essential for hypothalamic Pomc expression during embryogenesis (Nasif et al. 2015, Orquera et al. 2019). In addition, nPE1 contains a STAT3 consensus binding site (de Souza et al. 2005), suggesting a possible role for modulation of Pomc transcription by leptin-mediated JAK2/STAT3 signaling.
Leptin is a potent stimulator of Pomc neuron activity, but also plays vital roles during pregnancy. The ob/ob mouse is infertile, and administration of leptin restores fertility in both sexes (Chehab et al. 1996, Mounzih et al. 1997). Substantial increases of circulating leptin in the last trimester of pregnancy have been reported in animal models and humans (Tomimatsu et al. 1997, Anim-Nyame et al. 2000, Malik et al. 2005), which result from combinations of species’ specific differences in placenta-derived leptin (Masuzaki et al. 1997), increased fat mass, enhanced hormonal stimulation of leptin secretion from fat (such as insulin, estrogen and hCG), increased soluble leptin binding protein (Gavrilova et al. 1997) or other undetermined factors (Sivan et al. 1998, Zhao et al. 2003).
The current study showed that, at late pregnancy (dpc18.5), the Pomc∆1/∆1 and Pomcho∆1/∆1 dams exhibited a 2-fold and 2.3-fold higher level of circulating leptin, respectively, compared to the already elevated leptin levels of Pomc+/+dams (Figs 5A and 7F). Although Pomc∆1/∆1 dams had slightly, but not statistically higher fat mass compared to Pomc+/+dams at dpc18.5 (Pomc+/+: 3.07 ± 0.13 vs Pomc∆1/∆1: 3.90 ± 0.24 g), the 2-fold increase in circulating leptin cannot be simply attributed to ~0.9 g more fat mass in Pomc∆1/∆1 dams, indicating that the quantity of fat is not the major contributor for the exaggerated increase of circulating leptin in Pomc∆1/∆1 dams at the end of gestation. Concomitantly, we detected a 1.25-fold and 1.3-fold increase in LepRe expression in Pomc∆1/∆1 and Pomcho∆1/∆1 placentas, respectively (Figs 6 and 7E). Our lab has shown previously that deletion of both nPE1 and nPE2 from the Pomc gene results in obese, hyperleptinemic and infertile mice that exhibit low sympathetic tone, in general, and reduced renal sympathetic nerve activity, specifically (Chhabra et al. 2016, 2017). It has been demonstrated previously that inhibition of sympathetic activity on white adipocytes increases their leptin production (Rayner & Trayhurn 2001, Caron et al. 2018a). Since Pomc∆1/∆1 mice only maintain ~30% Pomc mRNA expression, it is likely that these mice also have decreased sympathetic output to adipose tissue, which leads to increased leptin release into the circulation.
Although some previous studies have reported reductions in ARC Pomc mRNA levels in pregnant or lactating rats, we did not observe any reductions from nonpregnant levels in our Pomc+/+ control dams at dpc18.5 and ppd10. Therefore, the increased expression of ARC Agrp without suppression of Pomc is apparently sufficient for mediating the hyperphagia that occurs during lactation. Future studies are necessary to confirm this hypothesis and to rule out additional neurochemical mechanisms described previously for hyperphagic pregnant rats (Ladyman et al. 2010). On the other hand, constitutive reduction of ARC Pomc expression by 70% in Pomc ∆1/∆1 dams led to a paradoxical decrease of fat mass during pregnancy with no effect on postnatal body weight, body composition or hyperphagia during lactation.
Intriguingly, we also detected a moderate increase of LepRe transcript abundance in the placentas of both Pomcho∆1/∆1 and Pomc∆1/∆1 dams compared to Pomc+/+ controls, although we failed to detect any further increase in circulating soluble leptin receptor protein in Pomc∆1/∆1 dams compared to Pomc+/+ controls. Because leptin levels were increased by roughly 20-fold from nonpregnant measurements to those at dpc18.5 in both genotypes of mice, while LEPRE levels only doubled, there was apparently no linear stoichiometry between the two molecules. Such nonlinearity has been reported previously, but not nearly to the extent of our data (Murakami et al. 2001).
One possible explanation for this discrepancy lies in fundamental characteristics of the ELISA itself that we used to measure soluble leptin receptor in plasma. Almost all of the earlier literature studying the complicated relationship between circulating leptin and soluble leptin receptor in pregnant mice was based on leptin radioimmunoassays and/or Western blots of leptin-like immunoreactivity after its affinity purification with Sepharose beads bound to recombinant leptin receptor (Gavrilova et al. 1997, Huang et al. 2001, Lammert et al. 2002, Yang et al. 2004). The soluble leptin receptor was quantified by Western blots after its affinity purification with Sepharose beads bound to recombinant leptin or was alternatively estimated from the binding capacity of serum to (125I) recombinant leptin (Gavrilova et al. 1997, Huang et al. 2001, Murakami et al. 2001). Collectively, these foundational studies concluded that 95% of circulating leptin in pregnant mice is bound to the soluble leptin receptor with high affinity; there is a 40-fold increase in the binding capacity of late-pregnancy maternal serum to leptin compared to serum from nonpregnant female mice; and the source of soluble leptin receptor in pregnant mice is mostly from translation of placental LepRe (Gavrilova et al. 1997). Moreover, it was shown that elevated soluble leptin receptor interfered with the accurate RIA measurement of leptin because of binding competition between the soluble leptin receptor and the leptin antisera during the immunoprecipitation to separate free from bound (125I) leptin tracer (Gavrilova et al. 1997).
To the best of our knowledge, the soluble leptin receptor, ELISA, we used is the only current commercially available assay that has been published by literature (Gan et al. 2012, Lee et al. 2018, Pereira et al. 2019, Yamada et al. 2019) , but has not been verified previously in studies using pregnant mice. Qualitatively, the assay was sufficient to confirm a significant increase in plasma soluble leptin receptor from dpc18.5 dams relative to nonpregnant females. However, the increase was only two-fold compared to typical 30 to 40-fold increases in other reports (Gavrilova et al. 1997, Lammert et al. 2002). Therefore, the ELISA may not have been sensitive enough to detect changes associated with the elevated levels of leptin in either genotype of mice at late pregnancy. Another possibility is that the LEPRE ELISA kit we used registers lower than expected values for soluble leptin receptor in plasma with greatly elevated leptin due to some unknown interference. However, data provided with the ELISA states that the addition of mouse leptin up to 50 ng/mL does not interfere with the assay. This does not exclude the possibility of assay interference from some other component of the plasma differentially produced in Pomc∆1/∆1 vs Pomc+/+ dams. Regardless of these considerations, further attempts to modify performance of the ELISA or to adopt alternative assays for quantifying either leptin or its soluble receptor are beyond the scope of this paper.
We evaluated the physiological importance of Pomc nPE1 on the placenta, and our results showed that lack of nPE1 did not change the expression of two critical placental genes (Plac1 and Tpbpa) or alter placental morphology based on intact layers of the labyrinth zone, junctional zone and maternal decidua basalis. Compared to Pomc expression in ARC, Pomc was expressed at a low but detectable level in placenta (Supplementary Fig. 3). Although nPE1 is a placental mammalian novelty, placental Pomc expression was unchanged in either Pomc∆1/∆1 or Pomcho∆1/∆1 mutant mice compared to WT mice. Therefore, similar to pituitary corticotrophs and melanotrophs, nPE1 apparently does not play a role in the regulation of Pomc expression in placenta. The lack of a prominent, altered phenotype in placentas or offspring of nPE1 deletion animals indicates that nPE1 and decreased ARC Pomc expression are not essential for pregnancy under ad libitum feeding conditions. Our previous work identified a rodent-specific deletion in the nPE1 core sequence, suggesting that nPE1 in mice may have different functional activities than in other mammals (Franchini et al. 2011). Moreover, laboratory mice were housed in ventilated cages without any external challenges such as food deprivation or extreme environmental conditions. The existence of nPE1 may facilitate the ability of placental animals to adapt to their environment for improved survival and reproduction, which happens during natural selection and is difficult to replicate using lab mice.
In conclusion, we have demonstrated that the neuronal Pomc enhancer nPE1, despite being a placental novelty of mammalian evolution, is dispensable for placental expression of Pomc during mouse pregnancy and for the marked metabolic adaptations of the dam during pregnancy and lactation under ad libitum access to food. Regardless of the unknown function of Pomc expression in placenta, we have revealed that a 70% reduction of Pomc expression in the ARC leads to substantial increases of plasma leptin and moderately high placental soluble leptin receptor transcript levels at late pregnancy, independent of total body fat mass, compared to control mice.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-19-0576.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by NIH grants R01-DK068400 (M J L and M R), U2C-DK110768 (M J L), K12-GM111725 (Z T), T32-NS076401 (G J) and T32-GM008322 (G J).
Acknowledgements
The authors thank Sarah Corral and Ian Gonzalez for preliminary studies, Talisha Sutton for mouse colony management and genotyping, Kavaljit Chhabra for monitoring and measuring body weight, Dr Nathan Qi and the Metabolism, Behavior and Bariatric Surgery Core of the Michigan Mouse Metabolic Phenotyping Center for assessing animal body composition and Dr Yiying Zhang from Columbia University for her advice on soluble leptin receptor measurement.
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