Abstract
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Central growth hormone action regulates metabolism during pregnancy
Abstract
The maternal organism undergoes numerous metabolic adaptations to become prepared for the demands associated with the coming offspring. These metabolic adaptations involve changes induced by several hormones that act at multiple levels, ultimately influencing energy and glucose homeostasis during pregnancy and lactation. Previous studies have shown that central growth hormone (GH) action modulates glucose and energy homeostasis. However, whether central GH action regulates metabolism during pregnancy and lactation is still unknown. In the present study, we generated mice carrying ablation of GH receptor (GHR) in agouti-related protein (AgRP)–expressing neurons, in leptin receptor (LepR)–expressing cells or in the entire brain to investigate the role played by central GH action during pregnancy and lactation. AgRP-specific GHR ablation led to minor metabolic changes during pregnancy and lactation. However, while brain-specific GHR ablation reduced food intake and body adiposity during gestation, LepR GHR knockout (KO) mice exhibited increased leptin responsiveness in the ventromedial nucleus of the hypothalamus during late pregnancy, although their offspring showed reduced growth rate. Additionally, both Brain GHR KO and LepR GHR KO mice had lower glucose tolerance and glucose-stimulated insulin secretion during pregnancy, despite presenting increased insulin sensitivity, compared with control pregnant animals. Our findings revealed that during pregnancy central GH action regulates food intake, fat retention, as well as the sensitivity to insulin and leptin in a cell-specific manner. Together, the results suggest that GH acts in concert with other “gestational hormones” to prepare the maternal organism for the metabolic demands of the offspring.
INTRODUCTION
Numerous metabolic adaptations prepare the maternal organism for the energy demands of the offspring. In this respect, pregnant animals commonly increase their food intake and accumulate body fat (5, 46, 62, 79, 82). Glucose homeostasis is also extensively altered during pregnancy, especially via the development of a transient insulin resistant state that is compensated by pancreatic beta-cell hyperplasia and increased glucose-stimulated insulin secretion (GSIS) capacity (6, 39, 73). A failure in the development of these typical adaptations produces important consequences in the mother (6, 14, 46, 82) and offspring (12, 69).
Much progress has been made in understanding the mechanisms involved in the metabolic adaptations during pregnancy. An important aspect is that these adaptations are likely driven by hormones highly secreted during pregnancy (5, 46). In rodents, early pregnancy is characterized by daily prolactin surges. However, pituitary prolactin secretion ceases because of increasing secretion of placental lactogens. Serum concentrations of leptin, estradiol, progesterone, and other placental hormones are also augmented along the pregnancy period (5). Several pieces of evidence indicate that prolactin plays a key role in the development of the neurobiological adaptations to pregnancy and lactation (33). For example, central prolactin injection reduces hypothalamic leptin sensitivity (3, 4, 60). Since leptin action in the brain inhibits food intake (68), the development of leptin resistance is a likely cause of the pregnancy-induced hyperphagia (2). Accordingly, ablation of the inhibitory protein suppressor of cytokine signaling 3 (SOCS3) in leptin receptor (LepR)–expressing cells restores leptin sensitivity and decreases food intake in pregnant mice (82). Leptin resistance during pregnancy may also involve impaired leptin transport from the systemic circulating into the brain (34, 65). Prolactin signaling in the pancreas is also required for the beta-cell hyperplasia observed during pregnancy (6, 39, 73). Thus, prolactin acts in multiple levels to prepare the maternal organism for the new demands associated with the offspring (33).
Although growth hormone (GH) has not been generally associated with the metabolic changes during pregnancy, several studies suggest that GH signaling is capable of affecting the maternal organism (7, 32, 50, 51). First, GH and prolactin share a similar intracellular signaling cascade, which is largely dependent on the activation of signal transducer and activator of transcription 5 (STAT5) proteins (19, 30, 75). Second, GH action during pregnancy is associated with some pathological conditions that impact the maternal metabolism as well as fetal growth and development (50). Third, humans produce a placental GH variant (GH-V), which is encoded by Gh2 gene, whereas Gh1 gene encodes the pituitary version of human GH. As seen with prolactin, the secretion of pituitary GH prevails during early pregnancy, whereas GH-V secretion increases from mid- to late pregnancy, suppressing pituitary GH secretion (50). However, it is worth mentioning that rodents do not possess a gene analogous to Gh2, so the role of placental GH in mice is uncertain. To uncover the effect of placental GH, Barbour et al. (7) generated transgenic mice that overexpressed the human GH-V. These mice presented higher body growth and developed severe insulin resistance (7). In another study, GH-V was infused from gestational days 12.5 to 18.5 in C57BL/6J mice (51). Although GH-V administration did not affect food intake or body weight in pregnant mice, reduced maternal insulin sensitivity was observed (51), suggesting a role of GH signaling in the development of the typical insulin resistance observed during pregnancy.
GH regulates several metabolic functions via its direct action in different organs and tissues, including pancreatic beta-cells (80), liver (25, 54), white adipose tissue (53), and skeletal muscle (55). The brain is also a direct target of GH action, as an extensive distribution of GH responsive neurons has been reported in well-known brain areas involved in the central regulation of metabolism (27, 29, 64, 83). In addition, many hypothalamic neurons that express the LepR are responsive to GH since a systemic GH injection induces STAT5 phosphorylation (pSTAT5) in these cells (13, 27, 31). As evidence that central GH action can affect energy metabolism, previous studies have shown that overexpression of GH in the central nervous system increases food intake and causes obesity (8, 84). Additionally, GH receptor (GHR) gene-disrupted or knockout (KO) mice become unresponsive to the orexigenic effect of ghrelin (22). Central GH injection increases the hypothalamic expression of orexigenic transcripts, such as Agrp and Npy, leading to higher food intake in mice (31). Moreover, GHR expression in agouti-related protein (AgRP)–expressing neurons is required for neuroendocrine and metabolic responses induced by food deprivation (31). Hence, GH action in specific neural populations is able to produce important metabolic consequences. However, to our knowledge no study has so far determined whether central GH action regulates metabolism during pregnancy and lactation. Therefore, the objective of the present study was to investigate the metabolic consequences during pregnancy and lactation of the ablation of GHR in AgRP neurons, in all LepR-expressing cells or in the entire brain. Our findings have the potential to provide novel insights toward the understanding of the hormonal factors involved in the gestational metabolic adaptations.
MATERIALS AND METHODS
Mice.
Genetic ablation of GHR was induced by initially breeding Ghrflox/flox mice (25, 54) with animals expressing the enzyme Cre recombinase under different gene enhancers/promoters: AgRP-IRES-Cre mouse (Agrptm1(cre)Lowl/J, The Jackson Laboratory), LepR-IRES-Cre mouse (B6.129-Leprtm2(cre)Rck/J, The Jackson Laboratory), or nestin-Cre mouse (B6.Cg-Tg(Nes-cre)1Kln/J, The Jackson Laboratory). The resulting heterozygous pups for the Ghrflox mutation and positive for Cre allele were further crossed with Ghrflox/flox mice, generating animals homozygous for the loxP-flanked Ghr allele and carrying the Cre gene. These conditional KO animals were crossed with Ghrflox/flox mice to produce the experimental animals. Thus, ~50% of the offspring was composed of Ghrflox/flox mice positive for the Cre allele (named as AgRP GHR KO, LepR GHR KO or Brain GHR KO mice), and the remaining Ghrflox/flox mice that were negative for the Cre allele became their respective control groups. For the histological experiments, AgRP-IRES-Cre, LepR-IRES-Cre, AgRP GHR KO, and LepR GHR KO mice were crossed with the Cre-inducible tdTomato reporter mouse [B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, The Jackson Laboratory] to induce the expression of the tdTomato fluorescent protein in AgRP or LepR cells. In these cases, control groups were composed of AgRP-IRES-Cre:Lox-Stop-Lox (LSL)-tdTomato or LepR-IRES-Cre::LSL-tdTomato mice, whereas conditional KO mice were also homozygous for the loxP-flanked Ghr allele. The mice were in the C57BL/6 background, weaned at 3–4 wk of age, and genotyped through PCR by using DNA extracted from the tail tip (REDExtract-N-Amp Tissue PCR Kit, Sigma). Mice were bred and maintained in standard conditions of light (12-h light/dark cycle) and temperature (22±
1°C). Mice received a regular rodent chow diet (2.99 kcal/g; 9.4% calories from fat). All experiments were carried out in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals and were previously approved by the Ethics Committee on the Use of Animals of the Institute of Biomedical Sciences at the University of São Paulo (protocol no. 73/2017).
Evaluation of GH responsive cells in the brain.
Adult nonpregnant mice received an acute intraperitoneal injection of GH from porcine pituitary (20 µg/g body weight, from Dr. A. F. Parlow, National Hormone and Peptide Program) and 90 min later were anesthetized with isoflurane and perfused with saline, followed by 10% buffered formalin solution. Brains were collected and postfixed in the same fixative for 1 h and then cryoprotected overnight at 4°C in 0.1 M PBS, pH 7.4, containing 20% sucrose. Brains were cut in frontal plane 30-µm-thick sections by using a freezing microtome. Brain slices were rinsed in 0.02 M potassium PBS, pH 7.4 (KPBS), followed by pretreatment in 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, slices were blocked in 3% normal donkey serum for 1 h, followed by incubation in anti-pSTAT5Tyr694 primary antibody (1:1,000; Cell Signaling; RRID: AB_2315225) for 40 h. After incubation in the primary antibody, sections were rinsed in KPBS and incubated for 90 min in AlexaFluor488-conjugated secondary antibody (1:500, Jackson ImmunoResearch). Sections were mounted onto gelatin-coated slides and covered with Fluoromount G mounting medium (Electron Microscopic Sciences, Hatfield, PA). The pSTAT5 was used as a marker of GH responsive cells, and pSTAT5 staining was co-localized with tdTomato reporter protein. A representative photomicrograph of leptin-responsive cells in the mediobasal hypothalamus was obtained from nonpregnant adult mice that received an acute intraperitoneal injection of mouse recombinant leptin (2.5 µg/g body weight; Dr. A. F. Parlow; National Hormone and Peptide Program). Brain sections were stained for phosphorylated STAT3 (pSTAT3), as described in the following section. 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).
Evaluation of leptin-responsive cells in the brain.
To determine central leptin sensitivity, mice on day 17 of pregnancy received a subcutaneous injection of sterile PBS or mouse recombinant leptin (2 µg/g body weight; Dr. A. F. Parlow, National Hormone and Peptide Program). After 3 h, mice were perfused and their brains processed to detect pSTAT3 since STAT3 is the major intracellular pathway recruited by leptin (66) and leptin-induced pSTAT3 staining has been used a marker of leptin responsiveness in the brain (2, 4, 47, 48, 61, 67, 70, 82). To label pSTAT3, brain slices were pretreated as described for pSTAT5 staining, except that sections were incubated in anti-pSTAT3Tyr715 primary antibody (1:1,000; RRID: AB_331586, Cell Signaling). Then, sections were incubated for 60 min in biotin-conjugated secondary antibody (1:1,000, Jackson ImmunoResearch), followed by 60 min in avidin-biotin complex (1:500, Vector Laboratories). The peroxidase reaction was performed using 0.05% 3,3′-diaminobenzidine, 0.25% nickel sulfate, and 0.03% hydrogen peroxide. The slides were covered with DPX mounting medium (Sigma, St. Louis, MO). The number of pSTAT3-positive cells in each area was plotted electronically using the ImageJ Cell Counter software (https://rsb.info.nih.gov/ij/). The leptin responsiveness was determined in one rostral-caudal level of the medial preoptic area (bregma: +0.50), two levels of the arcuate nucleus and ventromedial hypothalamus of the hypothalamus (bregma: −1.34 and −1.46), one level of the dorsomedial nucleus (bregma: −1.94), and one level of the ventral premammillary nucleus (bregma: −2.54). The Franklin and Paxinos mouse brain atlas was used as anatomical reference (26).
Evaluation of metabolic parameters in nonpregnant mice.
The metabolic parameters were initially determined in 8-wk-old sexually naive female mice. Food intake and body weight were recorded daily for 1 wk. Subsequently, mice were subjected to a glucose tolerance test (2 g glucose/kg sc) and an insulin tolerance test (1 IU insulin/kg sc). Then, a subgroup was euthanized to determine body length, femur length, and body adiposity via the measurements of the masses of the subcutaneous, periuterine, periovarian, and retroperitoneal fat pads. The deposits were collected bilaterally, and the values presented represent their average relativized to body weight.
Evaluation of metabolic parameters during pregnancy and lactation.
Sexually experienced males were introduced in the female’s cage. The males used to mate were always of the opposite genotype to the females. Thus, AgRP GHR KO, LepR GHR KO, and Brain GHR KO females were bred with Ghrflox/flox males, whereas control (Ghrflox/flox) females were bred with AgRP GHR KO, LepR GHR KO, and Brain GHR KO males. The presence of mating plugs was analyzed daily and its detection was considered as the first day of pregnancy. Females with copulatory plugs were put in individual cages and food intake and body weight were assessed daily during the entire pregnancy period (~19 days) and lactation (21 days). Additionally, the litter was standardized to five pups on the day of birth to ensure comparable metabolic demands during lactation. Litter growth was measured by weighting the offspring on days 1, 7, 14, and 21 of life. Changes in glucose homeostasis during pregnancy were determined in a different subgroup of mice that were subjected to a glucose tolerance test and an insulin tolerance test on days 14–15 and 17–18 of pregnancy, respectively. GSIS was determined on day 16–17 of pregnancy via blood samples collected to measure insulin levels at 0, 5, and 20 min after 2 g glucose/kg subcutaneous injection. On day 19 of pregnancy, mice were euthanized to collect serum samples and the hypothalamus, as well as to determine body adiposity, as previously described. Body adiposity was also determined in another group of mice at the end of the third week of lactation.
Hormonal dosage.
Serum samples of late-pregnant mice were used to determine leptin, insulin, and insulin-like growth factor-1 (IGF-1) levels by ELISA. Leptin (no. 90030, Crystal Chem) and insulin (no. 90080, Crystal Chem) ELISA kits have a detection limit of 0.2 and 0.1 ng/mL, respectively, and an intra- and interassay coefficient of variability ≤ 10%. IGF-1 ELISA kit (no. MG100, R&D Systems) has a detection limit of 3.5 pg/mL and an intra- and interassay coefficient of variability ≤ 6%.
Gene expression analysis.
Total RNA was extracted with TRIzol (Invitrogen). The Epoch Microplate Spectrophotometer (Biotek) was used to measure RNA quantity and quality. Total RNA was then incubated in DNase I RNase-free (Roche Applied Science), followed by reverse transcription using 2 µg of total RNA, the SuperScript II Reverse Transcriptase (Invitrogen) and random primers p(dN)6 (Roche Applied Science). Real-time PCR was performed using the 7500TM Real-Time PCR System (Applied Biosystems), Power SYBR Green or TaqMan Gene Expression PCR Master Mixes (Applied Biosystems), and specific primers for targeted genes (Table 1). Relative quantification of mRNA was calculated by 2-ΔΔCt. Data were normalized to the geometric average of Actb, Gapdh, and Ppia.
Table 1.
Primer sequences
| Target Gene | Forward Primer (5′-3′) | Reverse Primer (5′-3′) |
|---|---|---|
| Actb | gctccggcatgtgcaaag | catcacaccctggtgccta |
| Agrp | ctttggcggaggtgctagat | aggactcgtgcagccttacac |
| Cartpt | cagtcacacagcttcccgat | cagatcgaagcgttgcaaga |
| Cish | tcgggaatctgggtggtact | taggaatgtaccctccggca |
| Esr1 | gcagatagggagctggttca | tggagattcaagtccccaaa |
| Gapdh | gggtcccagcttaggttcat | tacggccaaatccgttcaca |
| Ghr (all isoforms) | atcaatccaagcctggggac | acagctgaatagatcctgggg |
| Hcrt | gacagcagtcgggcagag | ggcaccatgaactttccttc |
| Lepr (isoform b) | tgtcctactgctcggaacac | gctcaaatgtttcaggcttttgg |
| Npy | ccgcccgccatgatgctaggta | ccctcagccagaatgcccaa |
| Pomc | atagacgtgtggagctggtgc | gcaagccagcaggttgct |
| Prlr (all isoforms) | cagtaaatgccacgaacgaa | gaggaggctctggttcaaca |
| Socs1 | TaqMan Gene Expression Assay (Applied Biosystems) | |
| Socs3 | TaqMan Gene Expression Assay (Applied Biosystems) | |
Statistical analysis.
Data from control and AgRP GHR KO mice were analyzed by two-tailed Student’s t-test. The results obtained from control, Brain GHR KO, and LepR GHR KO mice were analyzed by one-way ANOVA and the Newman-Keuls multiple comparison post hoc test. Data of central leptin responsiveness were analyzed by two-way ANOVA and the Bonferroni multiple comparison post hoc test. All results were expressed as means±
SE. Statistical analyses were performed using GraphPad Prism software, considering only P values <0.05 as statistically significant.
RESULTS
Ablation of GHR in AgRP neurons causes no metabolic imbalances in nonpregnant mice.
In accordance with previous findings (31, 41), ~95% of AgRP neurons of the arcuate nucleus of the hypothalamus (ARH) contained pSTAT5 after a systemic injection of GH, indicating functional GHR expression in the majority of this neuronal population (Fig. 1A). To investigate the potential role of GH signaling in AgRP neurons for the metabolic changes that occur during pregnancy and lactation, mice carrying a specific ablation of Ghr gene only in AgRP-expressing cells were generated. As expected, AgRP neurons of the AgRP GHR KO mice became no longer responsive to GH injection, whereas pSTAT5 positive cells could still be observed in surrounding brain nuclei (Fig. 1B). Before evaluating AgRP GHR KO female mice during pregnancy and lactation, the energy and glucose homeostasis were initially determined in nonpregnant mice. As seen in male mice (31), AgRP GHR KO female mice showed similar body weight (Fig. 1C), body adiposity (Fig. 1D), daily food intake (Fig. 1E), glucose tolerance (Fig. 1, F and G), and insulin sensitivity (Fig. 1, H and I) compared with control animals. Thus, GHR ablation in AgRP neurons caused no metabolic imbalances in nonpregnant mice.
Ablation of growth hormone receptor (GHR) in agouti-related protein (AgRP) neurons causes no metabolic imbalances in nonpregnant mice. A and B: epifluorescence photomicrographs showing AgRP neurons (red) and growth hormone (GH)–induced pSTAT5 (green) in the arcuate nucleus of the hypothalamus (ARH) of nonpregnant mice. Yellow represents double-labeled cells. 3V, third ventricle; scale bar=
100 μm. C: mean body weight in control (n = 25) and AgRP GHR knockout (KO) (n = 10) mice. D: weight of adipose tissue deposits in control (n = 10) and AgRP GHR KO (n = 10) mice. E: mean daily food intake in control (n = 10) and AgRP GHR KO (n = 10) mice. F and G: blood glucose levels during a glucose tolerance test and the area under curve in control (n = 10) and AgRP GHR KO (n = 10) female mice. H and I: blood glucose levels during an insulin tolerance test and the area under curve.
AgRP-specific GHR ablation leads to minor metabolic changes during pregnancy and lactation.
Control and AgRP GHR KO female mice were mated with sexually experienced males. After the detection of the copulatory plug, these mice were single-housed to determine daily changes in body weight and food intake during pregnancy and the first 21 days of lactation. Pregnancy caused a robust increase in body weight (Fig. 2A) and weight gain (Fig. 2B) in both control and AgRP GHR KO mice. During lactation, the females maintained a body weight above pregestational values (Fig. 2A); however, AgRP GHR KO mice had significantly lower weight retention during most of the lactation period compared with control mice (Fig. 2B). Control and AgRP GHR KO mice showed an equivalent daily food intake during pregnancy, except for a few days in which AgRP GHR KO mice ingested a smaller amount of food (Fig. 2C). However, the cumulative food intake during the entire pregnancy or lactation period was not different between control and AgRP GHR KO mice (Fig. 2D). Additionally, no significant differences between groups were observed in body adiposity at late pregnancy (Fig. 2E) or after 21 days of lactation (Fig. 2F). Glucose homeostasis was also determined from mid- to late pregnancy. Pregnant control and AgRP GHR KO mice displayed a similar glucose tolerance (Fig. 3, A and B). However, pregnant AgRP GHR KO mice exhibited a slightly higher responsiveness to insulin, exhibiting a superior drop in blood glucose levels 90 min after insulin injection compared with pregnant control mice (Fig. 3C). However, the area under the curve of the insulin tolerance test was similar between the groups (Fig. 3D). The effects of AgRP-specific GHR ablation in the offspring were also determined. AgRP GHR KO mice gave birth to an equivalent number of pups compared with control dams (Fig. 3E). In addition, the offspring growth was similar between control and AgRP GHR KO mice (Fig. 3F).
Agouti-related protein (AgRP)–specific growth hormone receptor (GHR) ablation causes minor changes in energy balance during pregnancy and lactation. A and B: changes in body weight and in weight gain during pregnancy and lactation in control (n = 8) and AgRP GHR knockout (KO) (n = 10) mice. C and D: daily and cumulative food intake during pregnancy and lactation. E: weight of adipose tissue deposits on gestational day 18–19 in control (n = 8) and AgRP GHR KO (n = 4) mice. F: weight of adipose tissue deposits on the 21st day of lactation in control (n = 7) and AgRP GHR KO (n = 9) mice. *AgRP GHR KO is different from control (P < 0.05).
Agouti-related protein (AgRP)–specific growth hormone receptor (GHR) ablation causes minor changes in glucose homeostasis during pregnancy. A and B: blood glucose levels during a glucose tolerance test and the area under curve on gestational day 14–15 in control (n = 10) and AgRP GHR knockout (KO) (n = 5) mice. C and D: blood glucose levels during an insulin tolerance test and the area under curve on gestational day 17–18. E and F: litter size at birth and litter weight in 1-, 7-, 14-, and 21-day-old pups in control (n = 5–8) and AgRP GHR KO (n = 7–9) mice. *AgRP GHR KO is different from control (P < 0.05).
Brain- or LepR-specific GHR ablation affects growth, body adiposity, and food intake of nonpregnant mice.
GHR is widely expressed in the brain (29, 64, 83). In addition, several hypothalamic areas contain cells responsive to both GH (Fig. 4A) and leptin (Fig. 4B), including the arcuate nucleus of the hypothalamus (ARH) and the lateral hypothalamic area (LHA). In the ventromedial nucleus (VMH), GH responsive cells are distributed over the entire nucleus (Fig. 4A), whereas leptin-responsive cells are mainly restricted to its dorsomedial subdivision (VMHdm; Fig. 4B). To investigate the role of central GH action during pregnancy and lactation, mice carrying ablation of GHR in the entire brain (Brain GHR KO) or in LepR-expressing cells (LepR GHR KO) were studied. Brain-specific GHR ablation induced an 87% reduction in hypothalamic Ghr mRNA expression (0.13±
0.03 arbitrary units), compared with control animals on day 19 of pregnancy (1.00
±
0.20 arbitrary units; P = 0.002). GHR ablation only in LepR cells did not lead to significant changes in Ghr mRNA expression in the whole hypothalamus during pregnancy (1.18
±
0.16 arbitrary units; P = 0.6995). In control nonpregnant animals, a large amount of LepR-expressing cells co-expressed GH-induced pSTAT5, including neurons of the ARH (Fig. 4C) and VMHdm (Fig. 4D), as shown previously (13, 31). Nonpregnant Brain GHR KO mice exhibited virtually no GH-induced pSTAT5 in the ARH and VMH (Fig. 4, E and F), whereas LepR GHR KO mice only showed pSTAT5 in non-LepR-expressing cells (Fig. 4, G and H), demonstrating the cell-specific deletion of GHR. Regarding the metabolic consequences of GHR ablation in nonpregnant mice, Brain GHR KO females showed increased body weight (Fig. 5A), body length (Fig. 5C), and femur length (Fig. 5D) compared with control and LepR GHR KO mice. On the other hand, LepR GHR KO mice exhibited a similar body weight (Fig. 5A), body length (Fig. 5C), and femur length (Fig. 5D), but a significant reduction in body adiposity (Fig. 5B), whereas the relative food intake was increased compared with control and Brain GHR KO mice (Fig. 5E). No changes among the groups of nonpregnant mice were observed in glucose tolerance (Fig. 5, F and G) and insulin sensitivity (Fig. 5, H and I).
Brain- and leptin receptor (LepR)–specific growth hormone receptor (GHR) ablation. A and B: bright-field photomicrographs comparing the distribution of growth hormone (GH) and leptin-responsive cells in the mediobasal hypothalamus, identified, respectively, by pSTAT5 and pSTAT3 staining. C–H: epifluorescence photomicrographs showing LepR-expressing neurons (red) and growth hormone (GH)–induced pSTAT5 (green) in control (C and D), Brain GHR knockout (KO) (E and F) and LepR GHR KO (G and H) nonpregnant mice. Yellow represents double-labeled cells. 3V, third ventricle; ARH, arcuate nucleus; LHA, lateral hypothalamic area; VMH, ventromedial nucleus of the hypothalamus; VMHc, central subdivision of the VMH; VMHdm, dorsomedial subdivision of the VMH; VMHvl, ventrolateral subdivision of the VMH. Scale bars: A and B=
200 μm; C–H
=
100 μm.
Brain- and leptin receptor (LepR)–specific growth hormone receptor (GHR) ablation affects growth and body adiposity of nonpregnant mice. A: body weight in control (n = 8), Brain GHR knockout (KO) (n = 8), and LepR GHR KO (n = 8) mice. B: weight of adipose tissue deposits in control (n = 8), Brain GHR KO (n = 13), and LepR GHR KO (n = 20) mice. C and D: body and femur length in control (n = 9), Brain GHR KO (n = 14), and LepR GHR KO (n = 20) mice. E: daily food intake in control (n = 8), Brain GHR KO (n = 8), and LepR GHR KO (n = 8) mice. F and G: blood glucose levels during a glucose tolerance test and the area under curve in control (n = 8), Brain GHR KO (n = 8), and LepR GHR KO (n = 8) mice. H and I: blood glucose levels during an insulin tolerance test and the area under curve in control (n = 13), Brain GHR KO (n = 14), and LepR GHR KO (n = 17) mice. *Brain GHR KO is different from control (P < 0.05); †LepR GHR KO is different from control (P < 0.05); #LepR GHR KO is different from Brain GHR KO (P < 0.05).
GH signaling in the brain regulates metabolism during pregnancy and lactation.
Brain GHR KO mice maintained a significantly higher body weight throughout the pregnancy and lactation periods compared with control and LepR GHR KO females (Fig. 6A). Interestingly, Brain GHR KO mice also exhibited a higher weight gain in the last days of pregnancy in comparison with other groups (Fig. 6B). Although LepR GHR KO mice displayed a similar body weight and weight gain compared with control mice during pregnancy and lactation, GHR ablation in LepR cells induced an increase in food intake during part of the pregnancy and lactation periods (Fig. 6C). Consequently, the cumulative food intake during pregnancy and lactation was increased in LepR GHR KO mice compared with control and Brain GHR KO mice (Fig. 6D). In contrast, Brain GHR KO mice had a lower cumulative food intake during pregnancy compared with control mice (Fig. 6, C and D). At late pregnancy, Brain GHR KO mice exhibited the lowest subcutaneous adipose tissue weight (Fig. 6E). Other fat deposits were reduced in both Brain GHR KO and LepR GHR KO mice during late pregnancy compared with control animals (Fig. 6E). After 21 days of lactation, all groups exhibited equivalent body adiposity (Fig. 6F).
Growth hormone (GH) signaling in the brain regulates food intake and adiposity during pregnancy and lactation. A and B: changes in body weight and in weight gain during pregnancy and lactation in control (n = 14), Brain growth hormone receptor (GHR) knockout (KO) (n = 13), and leptin receptor (LepR) GHR KO (n = 12) mice. C and D: daily and cumulative food intake during pregnancy and lactation in control (n = 16), Brain GHR KO (n = 14), and LepR GHR KO (n = 12) mice. E: weight of adipose tissue deposits on gestational day 18–19 in control (n = 10), Brain GHR KO (n = 9), and LepR GHR KO (n = 10) mice. F: weight of adipose tissue deposits on the 21st day of lactation in control (n = 14), Brain GHR KO (n = 14), and LepR GHR KO (n = 9) mice. *Brain GHR KO is different from control (P < 0.05); †LepR GHR KO is different from control (P < 0.05); #LepR GHR KO is different from Brain GHR KO (P < 0.05).
Regarding glucose homeostasis during pregnancy, both Brain GHR KO and LepR GHR KO mice exhibited higher blood glucose levels during the glucose tolerance test, although the area under the curve was not statistically different among the groups (P = 0.0557; Fig. 7, A and B). The tendency toward a reduced glucose tolerance in Brain GHR KO and LepR GHR KO mice was associated with reduced GSIS (Fig. 7, C and D). Remarkably, Brain GHR KO and LepR GHR KO mice showed a significantly higher responsiveness to insulin during pregnancy, exhibiting reduced blood glucose levels during the insulin tolerance test compared with pregnant control mice (Fig. 7, E and F). In accordance with the increased insulin sensitivity, Brain GHR KO and LepR GHR KO mice had lower basal serum insulin levels at late pregnancy in comparison with control mice (Fig. 7G).
Brain growth hormone (GH) signaling regulates glucose homeostasis during pregnancy. A and B: blood glucose levels during a glucose tolerance test and the area under curve in control (n = 10), Brain growth hormone receptor (GHR) knockout (KO) (n = 10), and leptin receptor (LepR) GHR KO (n = 9) mice on gestational day 14–15. C and D: serum insulin levels during a glucose-stimulated insulin secretion test and the area under curve in control (n = 6), Brain GHR KO (n = 8), and LepR GHR KO (n = 6) mice on gestational day 15–16 day. E and F: blood glucose levels during an insulin tolerance test and the area under curve in control (n = 8), Brain GHR KO (n = 9), and LepR GHR KO (n = 7) mice on gestational day 17–18. G–I: serum insulin (G), leptin (H), and IGF-1 (I) levels on gestational day 18–19 in control (n = 8), Brain GHR KO (n = 9), and LepR GHR KO (n = 9) mice. J and K: litter size at birth and litter weight in 1-, 7-, 14-, and 21-day-old pups in control (n = 12–16), Brain GHR KO (n = 14), and LepR GHR KO (n = 9) mice. *Brain GHR KO is different from control (P < 0.05); †LepR GHR KO is different from control (P < 0.05); #LepR GHR KO is different from Brain GHR KO (P < 0.05).
Serum leptin levels during late pregnancy were significantly reduced in Brain GHR KO mice compared with other groups and in LepR GHR KO mice in comparison with control mice (Fig. 7H). In contrast, serum IGF-1 levels were increased during pregnancy only in Brain GHR KO mice compared with control and LepR GHR KO mice (Fig. 7I). An identical number of pups per litter was generated by control, Brain GHR K and LepR GHR KO mice (Fig. 7J). However, while the offspring growth of control and Brain GHR KO dams was similar, the offspring of LepR GHR KO females exhibited a significantly lower growth rate from the second week of life (Fig. 7K).
Increased leptin sensitivity in the ventromedial nucleus of pregnant LepR GHR KO mice.
To uncover possible neural mechanisms involved in the metabolic changes caused by central GHR ablation, hypothalamic gene expression was analyzed in late-pregnant mice. No significant changes among the groups were observed in the mRNA levels of Npy, Pomc, Cartpt, Hcrt, Esr1, Prlr (all isoforms), Lepr (isoform b), and Cish in the hypothalamus (Fig. 8A). However, LepR GHR KO mice exhibited increased hypothalamic mRNA expression of Agrp, Socs1, and Socs3 during late pregnancy compared with control and Brain GHR KO mice (Fig. 8A). Next, leptin responsiveness was evaluated in major hypothalamic areas that express the LepR (68). For this purpose, late-pregnant mice received an acute injection of PBS or leptin, and the number of neurons expressing pSTAT3 was evaluated in the areas of interest. As previously shown (61), leptin injection in pregnant mice, independently of the genotype, increased the number of pSTAT3 immunoreactive cells in all areas analyzed (Fig. 8B), including the medial preoptic area (Fig. 9, A–C), LHA, ARH (Fig. 9, D–F), VMH (Fig. 9, D–F), dorsomedial nucleus (Fig. 9, G–I), and ventral premammillary nucleus (Fig. 9, J–L) compared with PBS-injected mice (P < 0.001; two-way ANOVA). Notably, whereas Brain GHR KO mice showed similar leptin responsiveness in all hypothalamic areas compared with control animals, LepR GHR KO had increased number of pSTAT3 cells in the VMH in comparison with the other groups (Fig. 8B and Fig. 9, D–F).
Increased leptin sensitivity in the ventromedial nucleus of pregnant leptin receptor (LepR) growth hormone receptor (GHR) knockout (KO) mice. A: gene expression analysis in the hypothalamus of control (n = 8), Brain GHR KO (n = 8), and LepR GHR KO (n = 8) mice on gestational day 18–19. B: bar graphs comparing the number of pSTAT3 cells in different hypothalamic nuclei of PBS- or leptin-injected control (n = 4–7), Brain GHR KO (n = 4), and LepR GHR KO (n = 3–7) mice on gestational day 17. †LepR GHR KO is different from control (P < 0.05); #LepR GHR KO is different from Brain GHR KO (P < 0.05).
Leptin responsiveness in hypothalamic nuclei of late-pregnant mice. Representative photomicrographs of coronal brain sections showing pSTAT3 cells in the MPA (A–C), ARH (D–F), VMH (D–F), DMH (G–I) and PMV (J–L) of leptin-injected control (A, D, G, J), Brain growth hormone receptor (GHR) knockout (KO) (B, E, H, K) and leptin receptor (LepR) GHR KO (C, F, I, L) mice on gestational day 18–19. 3V, third ventricle; ARH, arcuate nucleus; DMH, dorsomedial nucleus of hypothalamus; LHA, lateral hypothalamic area; MPA, medial preoptic area; och, optic chiasm; ovlt, organum vasculosum of the lamina terminalis; VMH, ventromedial nucleus of the hypothalamus. Scale bar=
200 μm.
DISCUSSION
In the present study, we sought to determine whether central GH action is required for the occurrence of gestational metabolic adaptations (Fig. 10). We observed that central GH action regulates food intake, body adiposity, glucose homeostasis, and leptin sensitivity during pregnancy. Thus, our findings are in accordance with previous studies indicating that GH action during pregnancy regulates maternal metabolism (50). One key aspect is that, unlike humans, rodents do not produce the GH-V (40, 52). In spite of that, plasma GH levels are robustly elevated in late-pregnant mice and rats (23, 32, 77). However, the mechanisms associated with these increased circulating GH levels observed in pregnant rodents are unclear since decreased expression of Ghrh mRNA in the ARH and increased expression of somatostatin mRNA in the periventricular nucleus were observed in pregnant rats relative to virgin controls (23). Additionally, previous studies rule out an involvement of ghrelin-induced GH secretion during pregnancy and have shown evidence that GH is probably not secreted by the placenta (23, 77). Therefore, even though rodents do not possess a gene analogous to Gh2 and consequently cannot produce GH-V, GH is highly secreted in pregnant mice and the lack of GHR in the brain affects gestational metabolic adaptations.
Scheme that summarizes the present findings regarding the cell-specific actions of growth hormone (GH) regulating the metabolism during pregnancy.
AgRP neurons are an important neuronal population that controls food intake and are regulated by multiple hormonal inputs (1). In this sense, hormones that induce food intake, such as ghrelin, increase the activity of AgRP neurons, whereas anorexigenic hormones, including leptin, insulin, and cholecystokinin, inhibit AgRP neuronal activity (1, 68). In addition, the hyperphagia observed during pregnancy and lactation may involve AgRP neurons, since increased Agrp mRNA expression is found in the hypothalamus of pregnant (71, 74) and lactating rodents (15). Since most of the AgRP neurons express the GHR and GH is also able to activate these cells (31, 41), it is plausible to hypothesize that GH action on AgRP neurons could regulate food intake and body weight during gestation and lactation. We observed that AgRP GHR KO mice exhibited a slight decrease in food intake during gestation, a tendency toward increased insulin sensitivity at late pregnancy, and a reduced body weight along lactation. However, none of these effects were robust, indicating that GH action on AgRP neurons only plays minor effects in the gestational metabolic adaptations. Prolactin is known to be an important hormone involved in the metabolic changes during pregnancy and lactation (3, 5, 60). Interestingly, the metabolic effects of prolactin are also not mediated by AgRP neurons since these cells are not directly responsive to prolactin (49).
GH responsive cells are found in numerous brain nuclei (29, 64, 83). Of note, several hypothalamic neuronal populations that express the LepR are also responsive to GH (13, 27, 31). Given the importance of leptin signaling for the regulation of energy and glucose homeostasis (1, 68), including in the gestational period (58, 82), we also studied mice carrying GHR ablation in all LepR-expressing cells or in the entire brain. In these cases, growth or metabolic changes were already observed in nonpregnant females. Brain GHR KO mice clearly exhibited a phenotype of increased body growth as well as high circulating IGF-1 levels, probably caused by the loss of GH negative feedback in the hypothalamus. Accordingly, brain-specific GHR KO male mice exhibit increased Ghrh mRNA levels in the hypothalamus compared with wild-type mice (31). This phenotype contrasts with the mild hypopituitarism and reduced body weight exhibited by mice carrying the nestin-Cre only, since this transgene induces a central expression of human GH, which leads to reduction in pituitary GH secretion, secondary to the activation of negative feedback loops in the hypothalamus (18, 35). Thus, brain-specific GHR ablation prevents the neuroendocrine abnormalities exhibited by nestin-Cre transgenic mice. On the other hand, nonpregnant LepR GHR KO females showed increased food intake, but reduced body adiposity. These results suggest a possible increase in energy expenditure in LepR GHR KO mice. Neither brain- nor LepR-specific GHR ablation affected glucose homeostasis in nonpregnant female mice.
The apparent changes observed in nonpregnant Brain GHR KO and LepR GHR KO mice persisted during gestation and lactation. Thus, Brain GHR KO females maintained an elevated body weight throughout pregnancy and lactation, whereas LepR GHR KO mice continued to eat more food relative to body weight and maintained lower body fat than control animals at late pregnancy. Therefore, these differences are likely secondary to the phenotype of the animals, regardless of their reproductive state. However, Brain GHR KO mice showed lower food intake during pregnancy, leading to reduced fat retention. The reduced body fat in Brain GHR KO mice during late pregnancy was further supported by the decrease in serum leptin levels in comparison with control animals. These findings are in accordance with the orexigenic effect of central GH signaling (8, 31, 84) and with the fact that GH levels drastically increase during pregnancy (23, 32, 77). Thus, part of gestational hyperphagia may be caused by the orexigenic action of GH that is secreted during pregnancy. Since these effects were not observed in AgRP GHR KO or LepR GHR KO mice, our results indicate that non-LepR-expressing cells are behind the orexigenic effects of GH during pregnancy (Fig. 10). Although the specific brain area responsible for this effect remains to be determined, the ventrolateral subdivision of the VMH (VMHvl) is a possible candidate. Whereas LepR is mainly expressed in the VMHdm (Fig. 4B), the VMHvl contains cells responsive to GH (Fig. 4A), prolactin (10, 61), and sex steroids (17, 72). Additionally, a high co-expression between prolactin-induced pSTAT5 and estrogen receptor α (ERα) is observed in the VMHvl (28). Inactivation of ERα in the VMHvl leads to hyperphagia and obesity (59, 81), demonstrating the key role of VMHvl neurons for the regulation of energy balance.
Another important phenotype observed in pregnant Brain GHR KO and LepR GHR KO mice was associated with changes in glucose homeostasis, particularly the occurrence of glucose intolerance and reduced GSIS, together with increased insulin sensitivity. As glucose tolerance and insulin responsiveness were unchanged among the nonpregnant groups, these effects likely involve gestational metabolic adaptations. As mentioned earlier, overexpression or injection of GH-V causes insulin resistance in mice (7, 51). Since rodents do not express GH-V, the high secretion of the pituitary version of GH, especially during late pregnancy (23, 32, 77), may be associated with the gestational insulin resistance. It is well known that GH from pituitary also possesses potent diabetogenic effects and inhibits insulin action (19, 37, 38, 44, 45). Thus, our findings provide new evidence indicating that central GH action regulates insulin sensitivity during pregnancy, since ablation of GHR in the entire brain or in LepR-expressing cells protects mice from gestational insulin resistance (Fig. 10). Despite the increased insulin sensitivity, Brain GHR KO and LepR GHR KO mice exhibited lower glucose tolerance, which probably is related with a reduced GSIS capacity. The reasons for the lower GSIS capacity in pregnant Brain GHR KO and LepR GHR KO mice are unknown. It is possible that the increased insulin sensitivity caused by central GHR ablation simply reduced the need for the beta-cell hyperplasia that typically occurs during pregnancy (6, 39, 73). Thus, pregnant Brain GHR KO and LepR GHR KO mice are able to regulate glucose homeostasis even with a reduced GSIS capacity. The normal basal blood glucose level in these conditional KO mice is evidence in this regard. Another possibility is that GHR ablation may have occurred in pancreatic islets since both LepR and nestin are expressed in pancreatic islets (16, 20, 43, 57, 76, 85). Furthermore, it is well known that GHR regulates beta-cell function (56, 63, 78, 80). However, previous studies have shown that GHR ablation in LepR cells does not reduce Ghr mRNA levels in the pancreas (13), and nestin-expressing cells are not pancreatic endocrine cells that produce insulin, glucagon, somatostatin, or pancreatic polypeptide (20, 76, 85).
The litter growth of AgRP GHR KO and Brain GHR KO mice was normal, while the offspring of LepR GHR KO mice showed reduced growth rate compared with control animals. Since all litters were standardized to five pups at birth, the metabolic demands were equivalent among the experimental groups during lactation. Litter growth is influenced by maternal care and milk production. Although maternal behavior is mostly regulated by prolactin (11, 21), Bridges and Millard (9) showed that GH administration during lactation stimulates a faster onset of maternal behavior in steroid-treated rats. Thus, GH, like prolactin, can stimulate maternal behavior. Consequently, GHR ablation in specific neural populations could supposedly lead to impairment in the expression of maternal behavior. However, it is unclear why this phenotype was observed in LepR GHR KO mice but not in Brain GHR KO mice. Perhaps the deletion of GHR in the entire brain could have produced compensatory adaptations masking this particular effect. Another possibility is a decreased milk production in LepR GHR KO mice. An increased food intake during lactation is important to sustain an appropriate milk production (79). Since food intake of LepR GHR KO mice was higher during pregnancy and lactation, it is unlikely that changes in feeding were the limiting factor for the reduced litter growth of LepR GHR KO mice. The fat accumulated in pregnancy can be utilized during lactation to meet the high metabolic demand involved in milk production (62, 79). Since LepR GHR KO mice exhibited reduced pregestational and gestational body adiposity, the lower fat reserves may have been a factor that contributed to the reduction in their litter growth. It is also possible that GHR has been deleted from the mammary gland, since there is expression of LepR in this tissue (12). Although ablation of GHR can affect mammopoiesis and milk production (42), LepR expression is exclusively found in basal/myoepithelial cells of the mouse mammary gland, which are not the milk-secretory cells (12).
Previous studies have described the appearance of leptin resistance during pregnancy (2–4, 34, 58, 60, 82). The gestational leptin resistance seems to be cell specific and affects more significantly VMH neurons (4, 47, 48, 70, 82). Both GH and leptin-responsive neurons are found in the VMHdm, and approximately half of LepR-expressing cells in the VMHdm co-localize with GH-induced pSTAT5 (27, 31). Notably, GHR ablation in LepR-expressing cells increased leptin responsiveness in the VMH but not in other hypothalamic areas. The metabolic consequences of improving leptin responsiveness in the VMH are also unknown, especially because LepR GHR KO mice did not reduce food intake during pregnancy. Pregnant LepR GHR KO mice also exhibited increased hypothalamic mRNA levels of Agrp, Socs1, and Socs3. These changes seem to be counterintuitive in relation to the greater sensitivity to leptin observed in LepR GHR KO mice, since SOCS proteins, particularly SOCS3, inhibit leptin signaling (2, 82) and leptin action suppresses Agrp mRNA expression (1, 67, 68). However, leptin signaling induces SOCS expression; thus, the increased Socs1 and Socs3 expression in LepR GHR KO mice may actually indicate higher leptin responsiveness in the hypothalamus. From that perspective, earlier studies have used Socs3 expression as a marker of leptin-responsive cells (24, 36). Additionally, the upregulation of Agrp mRNA in the hypothalamus of LepR GHR KO mice may help to explain their increased food intake compared with the other groups.
In conclusion, our findings suggest that central GH action regulates energy and glucose homeostasis during pregnancy. More specifically, GH acts in LepR-expressing cells to modulate insulin sensitivity, whereas GH signaling in non-LepR neurons regulates the increases in food intake and fat retention during pregnancy (Fig. 10). Our findings, however, do not rule out the key role played by other hormones secreted during pregnancy (e.g., prolactin) or the possibility that GH exerts its effects via multiple tissues, but rather highlight the importance of GH action in the brain for the gestational adaptations that prepare the maternal organism for the metabolic demands of the coming offspring.
GRANTS
This study was supported by the São Paulo Research Foundation [FAPESP-Brazil, Grants 16/09679-4 (to I. Furigo), 17/02983-2 (to J. Donato), and 17/04006-4 (to G. Couto)], Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq-Brazil, Grant 160186/2015-3 (to P. Teixeira)] and National Institute on Aging Grant R01-AG-059779 (to J. Kopchick and E. List).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
P.D.S.T. and J.D. conceived and designed research; P.D.S.T., G.C.C., and I.C.F. performed experiments; P.D.S.T. and J.D. analyzed data; J.D. interpreted results of experiments; P.D.T. prepared figures; J.D. drafted manuscript; P.D.S.T., G.C.C., I.C.F., E.O.L., and J.J.K. edited and revised manuscript; P.D.S.T., G.C.C., I.C.F., E.O.L., J.J.K., and J.D. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank Ana Maria P. Campos for the technical assistance.
REFERENCES
Articles from American Journal of Physiology - Endocrinology and Metabolism are provided here courtesy of American Physiological Society
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Funding
Funders who supported this work.
HHS | National Institutes of Health (1)
Grant ID: R01AG059779
MCTI | Conselho Nacional de Desenvolvimento Científico e Tecnológico (1)
Grant ID: 160186/2015-3
NIA NIH HHS (1)
Grant ID: R01 AG059779
São Paulo Research Foundation (3)
Grant ID: 17/04006-4
Grant ID: 16/09679-4
Grant ID: 17/02983-2