Effect of high fat diet on maternal behavior, brain-derived neurotrophic factor and neural stem cell proliferation in mice expressing human placental lactogen during pregnancy.

Maternal obesity is a serious health concern because it increases risks of neurological disorders, including anxiety and peripartum depression. In mice, a high fat diet (HFD) in pregnancy can negatively affect placental structure and function as well as maternal behavior reflected by impaired nest building and pup-retrieval. In humans, maternal obesity in pregnancy is associated with reduced placental lactogen (PL) gene expression, which has been linked to a higher risk of depression. PL acting predominantly through the prolactin receptor maintains energy homeostasis and is a marker of placenta villous trophoblast differentiation during pregnancy. Impaired neurogenesis and low serum levels of brain-derived neurotrophic factor (BDNF) have also been implicated in depression. Augmented neurogenesis in brain during pregnancy was reported in the subventricular zone (SVZ) of mice at gestation day 7 and linked to increased prolactin receptor signaling. Here, we used transgenic CD-1 mice that express human (h) PL during pregnancy to investigate whether the negative effects of diet on maternal behavior are mitigated in these (CD-1[hGH/PL]) mice. Specifically, we examined the effect of a HFD on nest building prepartum and pup retrieval postpartum, as well as on brain BDNF levels and neurogenesis. In contrast to wild-type CD-1[WT]mice, CD-1[hGH/PL] mice displayed significantly less anxiety-like behavior, and showed no impairment in prepartum nest building or postpartum pup-retrieval when fed a HFD. Furthermore, the HFD decreased prepartum and increased postpartum BDNF levels in CD-1[WT] but not CD-1[hGH/PL] mice. Finally, neurogenesis in the SVZ as well as phosphorylated mitogen-activated protein kinase, indicative of lactogenic signaling, appeared unaffected by pregnancy and diet at gestation day 7 in CD-1[hGH/PL] mice. These observations indicate that CD-1[hGH/PL] mice are resistant to the negative effects of HFD reported for CD-1[WT] mice, including effects on maternal behaviors and BDNF levels, and potentially, neurogenesis. This difference probably reflects a direct or indirect effect of the products of the hGH/PL transgene.

Neurogenesis in the Maternal Rodent Brain: Impacts of Gestation-Related Hormonal Regulation, Stress, and Obesity.

In order to maintain maternal behavior, it is important that the maternal rodent brain promotes neurogenesis. Maternal neurogenesis is altered by the dynamic shifts in reproductive hormone levels during pregnancy. Thus, lifestyle events such as gestational stress and obesity that can affect hormone production will affect neuroendocrine control of maternal neurogenesis. However, there is a lack of information about the regulation of maternal neurogenesis by placental hormones, which are key components of the reproductive hormonal profile during pregnancy. There is also little known about how maternal neurogenesis can be affected by health concerns such as gestational stress and obesity, and its relationship to peripartum mental health disorders. This review summarizes the changing levels of neurogenesis in mice and rats during gestation and postpartum as well as regulation of neurogenesis by pregnancy-related hormones. The influence of neurogenesis on maternal behavior is also discussed while bringing attention to the effect of health-related concerns during gestation, such as stress and obesity on neuroendocrine control of maternal neurogenesis. In doing so, this review identifies the gaps in the literature and specifically emphasizes the importance of further research on maternal brain physiology to address them.

Elimination of endogenous high molecular weight FGF2 prevents pressure-overload-induced systolic dysfunction, linked to increased FGFR1 activity and NR1D1 expression.

Fibroblast growth factor 2 (FGF2), produced as high (Hi-) and low (Lo-) molecular weight isoforms, is implicated in cardiac response to injury. The role of endogenous FGF2 isoforms during chronic stress is not well defined. We investigated the effects of endogenous Hi-FGF2 in a mouse model of simulated pressure-overload stress achieved by transverse aortic constriction (TAC) surgery. Hi-FGF2 knockout mice, expressing only Lo-FGF2, FGF2(Lo), and wild-type mice, FGF2(WT), expressing both Hi-FGF2 and Lo-FGF2, were used. By echocardiography, a decline in systolic function was observed in FGF2(WT) but not FGF2(Lo) mice compared to corresponding sham-operated animals at 4-8 weeks post-TAC surgery. TAC surgery increased markers of myocardial stress/damage including B-type natriuretic peptide (BNP) and the pro-cell death protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 (Bnip3) in FGF2(WT) but not FGF2(Lo) mice. In FGF2(Lo) mice, cardiac levels of activated FGF receptor 1 (FGFR1), and downstream signals, including phosphorylated mTOR and p70S6 kinase, were elevated post-TAC. Finally, NR1D1 (nuclear receptor subfamily 1 group D member 1), implicated in cardioprotection from pressure-overload stress, was downregulated or upregulated in the presence or absence, respectively, of Hi-FGF2 expression, post-TAC surgery. In wild-type cardiomyocyte cultures, endothelin-1 (added to simulate pressure-overload signals) caused NR1D1 downregulation and BNP upregulation, similar to the effect of TAC surgery on the FGF2(WT) mice. The NR1D1 agonist SR9009 prevented BNP upregulation, simulating post-TAC findings in FGF2(Lo) mice. We propose that elimination of Hi-FGF2 is cardioprotective during pressure-overload by increasing FGFR1-associated signaling and NR1D1 expression.

A potential role for insulin treatment during pregnancy in reducing postpartum psychological distress in maternal obesity: an administrative population health study.

BACKGROUND: Studies have found an association between obesity and an increased risk for peripartum depression, which has also been linked to decreased placental lactogen levels. In addition, women with obesity treated for gestational diabetes with insulin were found to have increased levels of placental lactogen. Treatment options exist for perinatal and postpartum depression however they pose a risk to the developing offspring. Thus, prevention as well as markers for early identification of peripartum depression are needed. Therefore, our study objective is to identify the association between insulin treatment in pregnancy and the risk of postpartum psychological distress (abbreviated here as PPD) among cohorts of women with and without obesity. METHODS: Administrative health data (2002/03-2018/19) were used to identify a cohort of women (age 15+ years) who gave birth (N = 250,746) and had no pre-existing mood/anxiety disorders or diabetes (N = 222,863 excluded). Women were then divided into two groups: lean (N = 17,975) and with obesity (N = 9908), which was identified by a recorded maternal weight of > 38 to < 65.6 kg and ≥ 85 to < 186 kg (respectively). The risk of PPD within one year after delivery with and without insulin treatment was assessed by Poisson regression analysis. Models were adjusted for maternal age group (at pregnancy start date) and area-level income (at delivery). RESULTS: The unadjusted risk of PPD was higher in the obesity group (8.56%; 95% CI 8.00-9.15) than in the lean group (6.93%; 95% CI 6.56-7.33). When no insulin treatment was given during pregnancy, mothers with obesity had a significantly higher risk of PPD than the lean group (aRR 1.27; 95% CI 1.17-1.39; p < 0.0001). However, when women with obesity and insulin treatment were compared to the lean group with no insulin treatment, no significant difference in the risk of PPD was observed between the groups (aRR 1.30; 95% CI 0.83-2.02; p = 0.248). CONCLUSION: This is the first study to demonstrate a positive association between insulin treatment in pregnancy among women with obesity and reduced PPD rates, suggesting insulin as a possible preventative measure. However, the biological mechanism behind the observed positive effect of insulin on PPD rates remains to be investigated.

Dexamethasone Rescues an Acute High-Fat Diet-Induced Decrease in Human Growth Hormone Gene Expression in Male Partially Humanized CD-1 Mice.

Obesity in puberty, already a time of insulin resistance, increases the risk of developing type 2 diabetes. Human (h) growth hormone (GH) levels also peak during puberty, where it contributes to growth and energy homeostasis through positive effects on maintaining pancreatic ß cell mass. Thus, it is important to understand the effects of overeating and obesity on hGH production in puberty. Three days of overeating in young male adults or high-fat diet (HFD) in pubescent male transgenic (171hGH/CS) CD-1 mice containing the hGH gene (hGH-N) results in excess insulin and a decrease in hGH production. This reduction in these mice occurred during the light phase of the daily cycle, and was associated with decreased availability of the clock-related transcription factor Brain and Muscle ARNT-Like 1 (Bmal1). However, the HFD-induced decrease in hGH-N expression was blocked by forced daily swim activity, which is expected to increase glucocorticoid (GC) levels. The aim of the study was to assess whether GCs, specifically daily injections with a pharmacological dose of dexamethasone (DEX) in the light or dark phase of the daily cycle, can limit the negative effect of HFD for 3 days on hGH-N expression in male 171hGH/CS mice. DEX treatment increased or rescued hGH-N RNA levels, and was associated with elevated Bmal1 transcripts when assessed 12 h after final treatment, and at a time when serum corticosterone levels were suppressed >90%. In addition, a diet-dependent effect on hGH-N RNA levels was observed at 36 h after final treatment, but only in the light stage, presumably due to residual effects of DEX treatment and/or recovery of endogenous corticosterone levels. This is the first evidence for a direct effect of GCs on hGH-N expression in vivo and the ability to potentially limit the negative effect of overeating/obesity on hGH production in puberty.

Effects of high fat diet-induced obesity and pregnancy on prepartum and postpartum maternal mouse behavior.

Obesity before and during pregnancy negatively affects the mental and physical health of the mother. A diet high in fat also increases the risk for anxiety, depression and cognitive dysfunction. We examined the effects of high fat diet (HFD) -induced obesity and pregnancy on maternal behavior, cognitive function and anxiety- and depression-like behaviors in mice. Four-week-old female CD-1 mice were placed on a HFD or regular chow diet (RCD) for 5 weeks. Mice were maintained on either diet as non-pregnant HFD and RCD groups, or allowed to breed, and then fed these diets throughout gestation, lactation and after weaning, as pregnant HFD and RCD groups. Mice on HFD but not on RCD for 5 weeks pre-pregnancy significantly gained weight and had impaired glucose clearance. Maternal behavior was assessed by nest building prepartum and pup-retrieval postpartum. Anxiety-like behavior was evaluated both prepartum and postpartum by elevated plus maze and cognitive function was assessed by the novel object recognition test postpartum. Anhedonia, a measure of impaired reward function, is an endophenotype of depression and was assessed by sucrose preference test pre- and post-weaning in dams. Mice on HFD in pregnancy exhibited both impaired maternal behavior and cognitive function in the postpartum period. We did not detect measurable differences between the HFD and RCD groups in anxiety-like behavior in the prepartum period. In contrast, HFD was also associated with anhedonia in pregnant mice pre-weaning, and anxiety-like behavior post-weaning. Thus, HFD has a negative effect on maternal behavior in the outbred CD-1 mouse, which provides a model to study associated outcomes and related mechanisms.

Sleep deprivation and diet affect human GH gene expression in transgenic mice in vivo.

Human (h) growth hormone (GH) production studies are largely limited to effects on secretion. How pituitary hGH gene (hGH-N/GH1) expression is regulated is important in our understanding of the role hGH plays in physiology and disease. Here we assess for the first time the effect of sleep deprivation (SD) and high-fat diet (HFD) on hGH-N expression in vivo using partially humanized 171hGH/CS transgenic (TG) mice, and attempted to elucidate a role for DNA methylation. Activation of hGH-N expression requires interactions between promoter and upstream locus control region (LCR) sequences including pituitary-specific hypersensitive site (HS) I/II. Both SD and diet affect hGH secretion, but the effect of SD on hGH-N expression is unknown. Mice fed a HFD or regular chow diet for 3 days underwent SD (or no SD) for 6 h at Zeitgeber time (ZT) 3. Serum and pituitaries were assessed over 24 h at 6-h intervals beginning at ZT 14. SD and HFD caused significant changes in serum corticosterone and insulin, as well as hGH and circadian clock-related gene RNA levels. No clear association between DNA methylation and the negative effects of SD or diet on hGH RNA levels was observed. However, a correlation with increased methylation at a CpG (cytosine paired with a guanine) in a putative E-box within the hGH LCR HS II was suggested in situ. Methylation at this site also increased BMAL1/CLOCK-related nuclear protein binding in vitro. These observations support an effect of SD on hGH synthesis at the level of gene expression.

Obesity and regulation of human placental lactogen production in pregnancy.

The four genes coding for placental members of the human (h) growth hormone (GH) family include two that code independently for placental lactogen (PL), also known as chorionic somatomammotrophin hormone, one that codes for placental growth hormone (PGH) and a pseudogene for which RNA but no protein product is reported. These genes are expressed preferentially in the villus syncytiotrophoblast of the placenta in pregnancy. In higher primates, the placental members, including hPL and PGH, are the result of multiple duplication events of the GH gene. This contrasts with rodents and ruminants, where PLs result from duplication of the prolactin (PRL) gene. Thus, unlike their mouse counterparts, the hPL and PGH hormones bind both lactogenic and somatogenic receptors with varying affinity. Roles influenced by nutrient availability in both metabolic control in pregnancy and maternal behaviour are supported. However, the effect maternal obesity has on the activation of placental members of the hGH gene family, particularly the expression and function of those genes, is poorly understood. Evidence from partially humanised hGH/PL transgenic mice indicates that both the remote upstream hPL locus control region (LCR) and more gene-related regulatory regions are required for placental expression in vivo. Furthermore, a specific pattern of interactions between the LCR and hPL gene promoter regions is detected in term placenta chromatin from women with a normal body mass index (BMI) in the range 18.5-25 kg m-2 by chromosome conformation capture assay. This pattern is disrupted with maternal obesity (class II BMI > 35 kg m-2 ) and associated with a > 40% decrease in term hPL RNA levels, as well as serum hPL but not PRL levels, during pregnancy. The relative importance of the chromosomal architecture and predicted properties for transcription factor participation in terms of hPL production and response to obesity are considered, based on comparison with components required for efficient human pituitary GH gene expression.

Negative Effects of Cyclic Palmitate Treatment on Glucose Responsiveness and Insulin Production in Mouse Insulinoma Min6 Cells Are Reversible.

Pancreatic ß-cell failure is characterized by compromised insulin secretion in response to glucose, which ultimately results in hyperglycemia, the clinical hallmark of type 2 diabetes mellitus (T2DM). Acute exposure to plasma free fatty acids (FFAs) potentiates glucose stimulated insulin secretion (GSIS), while chronic exposure impairs GSIS, and the latter has been associated with the mechanism of ß cell failure in obesity linked T2DM. By contrast, growth hormone (GH) signaling has been linked positively to GSIS in ß cells. Numerous studies have examined chronic exposure of ß cells to elevated FFAs both with in vivo cohorts and in vitro models. Little attention, however, has been given to the fluctuation of plasma FFA levels due to rhythmic effects that are affected by daily diet and fat intake. Mouse insulinoma Min6 cells were exposed to cyclic/daily palmitate treatment over 2 and 3 days to assess effects on GSIS. Cyclic/daily palmitate treatment with a period of recovery negatively affected GSIS in a dose-dependent manner. Removal of palmitate after two cycles/day resulted in reversal of the effect on GSIS, which was also reflected by relative gene expression involved in insulin biosynthesis (Ins1, Ins2, Pdx1, and MafA) and GSIS (glucose 2 transporter and glucokinase). Modest positive effects on GSIS and glucokinase transcript levels were also observed when Min6 cells were cotreated with human GH and palmitate. These observations indicate that like continuous palmitate treatment, cyclic exposure to palmitate can acutely impair GSIS over 48 and 72 h. However, they also suggest that the negative effects of short periods of exposure to FFAs on ß cell function remain reversible.

Elimination or neutralization of endogenous high-molecular-weight FGF2 mitigates doxorubicin-induced cardiotoxicity.

Cardiac fibroblast growth factor 2 (FGF2) exerts multiple paracrine activities related to cardiac response to injury. Endogenous FGF2 is composed of a mixture of 70% high- and 30% low-molecular-weight isoforms (Hi-FGF2 and Lo-FGF2, respectivley); although exogenously added Lo-FGF2 is cardioprotective, the roles of endogenous Hi-FGF2 or Lo-FGF2 have not been well defined. Therefore, we investigated the effect of elimination of Hi-FGF2 expression on susceptibility to acute cardiac damage in vivo caused by an injection of the genotoxic drug doxorubicin (Dox). Mice genetically depleted of endogenous Hi-FGF2 and expressing only Lo-FGF2 [FGF2(Lo) mice] were protected from the Dox-induced decline in ejection fraction displayed by their wild-type FGF2 [FGF2(WT)] mouse counterparts, regardless of sex, as assessed by echocardiography for up to 10 days post-Dox treatment. Because cardiac FGF2 is produced mainly by nonmyocytes, we next addressed potential contribution of fibroblast-produced FGF2 on myocyte vulnerability to Dox. In cocultures of neonatal rat cardiomyocytes (r-cardiomyocytes) with mouse fibroblasts from FGF2(WT) or FGF2(Lo) mice, only the FGF2(Lo)-fibroblast cocultures protected r-cardiomyocytes from Dox-induced mitochondrial and cellular damage. When r-cardiomyocytes were cocultured with or exposed to conditioned medium from human fibroblasts, neutralizing antibodies for human Hi-FGF-2, but not total FGF2, mitigated Dox-induced injury of cardiomyocytes. We conclude that endogenous Hi-FGF2 reduces cardioprotection by endogenous Lo-FGF2. Antibody-based neutralization of endogenous Hi-FGF2 may offer a prophylactic treatment against agents causing acute cardiac damage. NEW & NOTEWORTHY Cardiomyocytes, in vivo and in vitro, were protected from the deleterious effects of the anticancer drug doxorubicin by the genetic elimination or antibody-based neutralization of endogenous paracrine high-molecular-weight fibroblast growth factor 2 isoforms. These findings have a translational potential for mitigating doxorubicin-induced cardiac damage in patients with cancer by an antibody-based treatment.

Cardiac Fgf-16 Expression Supports Cardiomyocyte Survival and Increases Resistance to Doxorubicin Cytotoxicity.

The fibroblast growth factor (FGF) 16 gene is preferentially expressed by cardiomyocytes after birth with levels increasing into adulthood. Null mice and isolated heart studies suggest a role for FGF-16 in cardiac maintenance and survival, including increased resistance to doxorubicin (DOX)-induced injury. A single treatment with DOX was also shown to rapidly deplete endogenous rat FGF-16 mRNA at 6 h in both adult heart and neonatal cardiomyocytes. However, the effect of DOX on rat cardiac function at the time of decreased FGF-16 gene expression and the effect of FGF-16 availability on cardiomyocyte survival, including in the context of acute DOX cytotoxicity, have not been reported. The objective was to assess the effect of acute (6 and 24 h) DOX treatment on cardiac function and the effects of FGF-16 small interfering RNA "knockdown," as well as adenoviral overexpression, in the context of acute DOX cytotoxicity, including cardiomyocyte survival and DOX efflux transport. A significant decrease in heart systolic function was detected by echocardiography in adult rats treated with 15 mg DOX/kg at 6 h; however, unlike FGF-16, there was no change in atrial natriuretic peptide transcript levels. Both systolic and diastolic dysfunctions were observed at 24 h. In addition, specific FGF-16 "knockdown" in neonatal rat cardiomyocytes results in a significant increase in cell death. Conversely, adenoviral FGF-16 overexpression was associated with a significant decrease in cardiomyocyte injury as a result of 1 µM DOX treatment. A specific increase in efflux transporter gene expression and DOX efflux was also seen, which is consistent with a reduction in DOX cytotoxicity. Finally, the increased efflux and decreased DOX-induced damage with FGF-16 overexpression were blunted by inhibition of FGF receptor signaling. These observations are consistent with FGF-16 serving as an endogenous cardiomyocyte survival factor, which may involve a positive effect on regulating efflux transport to reduce cardiotoxicity.

A useful model to compare human and mouse growth hormone gene chromosomal structure, expression and regulation, and immune tolerance of human growth hormone analogues.

Human (h) pituitary growth hormone (GH) is both physiologically and clinically important. GH reaches its highest circulatory levels in puberty, where it contributes to energy homeostasis and somatogenic growth. GH also helps to maintain tissues and organs and, thus, health and homeostasis. A reduction in the rate of hGH production begins in middle age but if GH insufficiency occurs this may result in tissue degenerative and metabolic diseases. As a consequence, hGH is prescribed under conditions of GH deficiency and, because of its lipolytic activity, stimulation of hGH release has also been used to treat obesity. However, studies of normal GH production and particularly synthesis versus secretion are not feasible in humans as they require sampling normal pituitaries from living subjects. Furthermore, human (or primate) GH structure and, as such, regulation and potential function, is distinct from non-primate rodent GH. As a result, most information about hGH regulation comes from measurements of secreted levels of GH in humans. Thus, partially humanized hGH transgenic mice, generated containing fragments of human chromosome 17 that include the intact hGH gene locus and many thousands of flanking base pairs as well as the endogenous mouse (m) GH gene provide a potentially useful model. Here we review this mouse model in terms of its ability to allow comparison of hGH versus mGH gene expression, and specifically: (i) GH locus structure as well as regulated and rhythmic expression; (ii) their ability to model a clinical assessment of hGH production in response to overeating and hyperinsulinemia as well as a possible effect of exercise, and (iii) their hGH-related immune tolerance and thus potential for testing hGH-related analogue immunogenicity.

Chromosomal architecture and placental expression of the human growth hormone gene family are targeted by pre-pregnancy maternal obesity.

The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this "resistance", and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS ( hCS-A and hCS-B) and placental growth hormone ( hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene ( hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III-V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placenta-specific chromosomal architecture, including interaction between HS III-V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m2) by using the chromosome conformation capture assay. This architecture was disrupted by pre-pregnancy maternal obesity (BMI >35 kg/m2), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-ß association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS.

Fibroblast growth factor-2-mediated protection of cardiomyocytes from the toxic effects of doxorubicin requires the mTOR/Nrf-2/HO-1 pathway.

BACKGROUND: Cardiotoxic side effects impose limits to the use of anti-tumour chemotherapeutic drugs such as doxorubicin (Dox). There is a need for cardioprotective strategies to prevent the multiple deleterious effects of Dox. Here, we examined the ability of administered fibroblast growth factor-2 (FGF-2), a cardioprotective protein that is synthesized as high and low molecular weight (Hi-, Lo-FGF-2) isoforms, to prevent Dox-induced: oxidative stress; cell death; lysosome dysregulation; and inactivation of potent endogenous protective pathways, such as the anti-oxidant/detoxification nuclear factor erythroid-2-related factor (Nrf-2), heme oxygenase-1 (HO-1) axis. METHODS AND RESULTS: Brief pre-incubation of neonatal rat cardiomyocyte cultures with either Hi- or Lo-FGF-2 reduced the Dox-induced: oxidative stress; apoptotic/necrotic cell death; lysosomal dysregulation; decrease in active mammalian target of Rapamycin (mTOR). FGF-2 isoforms prevented the Dox-induced downregulation of Nrf-2, and promoted robust increases in the Nrf-2-downstream targets including the cardioprotective protein HO-1, and p62/SQSTM1, a multifunctional scaffold protein involved in autophagy. Chloroquine, an autophagic flux inhibitor, caused a further increase in p62/SQSTM1, indicating intact autophagic flux in the FGF-2-treated groups. A selective inhibitor for HO-1, Tin-Protoporphyrin, prevented the FGF-2 protection against cell death. The mTOR inhibitor Rapamycin prevented FGF-2 protection, and blocked the FGF-2 effects on Nrf-2, HO-1 and p62/SQSTM1. CONCLUSIONS: In an acute setting Hi- or Lo-FGF-2 protect cardiomyocytes against multiple Dox-induced deleterious effects, by a mechanism dependent on preservation of mTOR activity, Nrf-2 levels, and the upregulation of HO-1. Preservation/activation of endogenous anti-oxidant/detoxification defences by FGF-2 is a desirable property in the setting of Dox-cardiotoxicity.

Expression of the Cardiac Maintenance and Survival Factor FGF-16 Gene Is Regulated by Csx/Nkx2.5 and Is an Early Target of Doxorubicin Cardiotoxicity.

The fibroblast growth factor (FGF) 16 gene (Fgf-16) is preferentially expressed by neonatal cardiomyocytes after birth, with levels increasing into adulthood. Null mice and isolated heart studies suggest a role for FGF-16 in cardiac maintenance and survival, including increased resistance to doxorubicin (DOX)-induced injury. However, the effect of DOX on endogenous FGF-16 synthesis and specifically regulation of cardiac Fgf-16 expression has not been reported. Here we assess the effect of DOX on FGF-16 RNA levels and stability as well as promoter activity and use sequence analysis, knockdown, and overexpression to investigate the role of cardiac transcription factor(s) implicated in the response. Endogenous FGF-16 RNA levels were reduced >70% in 8-week-old rats treated with 15 mg DOX/kg for 6 h. This was modeled in neonatal rat cardiomyocyte cultures, where an equivalent decrease was also seen within 6 h of 1 µM DOX treatment. Six kilobases of mouse Fgf-16 upstream flanking and promoter DNA was also assessed for DOX responsiveness in transfected cardiomyocytes. A decrease in FGF-16 promoter activity was seen with only 747 base pairs containing the Fgf-16 TATA box that includes a putative and highly conserved binding site for the cardiac transcription factor Csx/Nkx2.5. There was also no effect of DOX on FGF-16 RNA stability, consistent with transcriptional control. Levels and binding of Csx/Nkx2.5 to the FGF-16 promoter were reduced with DOX treatment. Knockdown of Csx/Nkx2.5 specifically decreased endogenous FGF-16 RNA and protein levels, whereas Csx/Nkx2.5 overexpression stimulated levels, and increased resistance to the rapid DOX-induced depletion of FGF-16. These observations indicate that Fgf-16 expression is directly regulated by Csx/Nkx2.5 in neonatal cardiomyocytes, and a negative effect of DOX on Csx/Nkx2.5 and, thus, endogenous FGF-16 synthesis may contribute indirectly to its cardiotoxic effects. Targeting FGF-16 levels could, however, offer increased resistance to cardiac injury.

Evidence for a Circadian Effect on the Reduction of Human Growth Hormone Gene Expression in Response to Excess Caloric Intake.

Rhythmicity of biological functions is fundamental for optimal adaptations to environmental cues. Growth hormone (GH) is a major metabolic homeostatic factor that is secreted with a circadian pattern, but whether it is synthesized rhythmically is unknown. We used transgenic mice containing the human (h) GH gene (hGH1) locus to investigate the rhythmicity of hGH synthesis and secretion and to show that RNA and secreted protein levels oscillate over a 24-h cycle. Analysis of hGH1 promoter sequences revealed an enhancer motif (E-box) element that binds the circadian transcriptional machinery (Bmal1 and Clock). Furthermore, Bmal1/Clock were able to transactivate the hGH1 promoter, and mutation of this E-box element adversely affected basal activity after gene transfer. The ability of Bmal1 to bind the hGH1 promoter region containing the E-box element was confirmed in the hGH1 transgenic mouse pituitary in situ Occupancy was reduced in mice fed a high fat diet during the light (inactive) stage of the daily cycle in mice and corresponded to a decrease in hGH1 RNA levels. The decreases in occupancy and RNA levels were not seen, however, during the dark (active) stage. A chromatin loop required for efficient postnatal hGH1 expression was negatively affected by the high fat diet in the light but not dark stage similar to the pattern observed with Bmal1 association with the promoter region. This is the first evidence that hGH synthesis follows a diurnal rhythm and of dynamic associations of the circadian machinery with a component of a chromosomal structure of the hGH1 locus that is essential for efficient expression.

Expression of Placental Members of the Human Growth Hormone Gene Family Is Increased in Response to Sequential Inhibition of DNA Methylation and Histone Deacetylation.

The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V, are located at a single locus on chromosome 17. Efficient expression of these placental genes has been linked to local regulatory (5' P and 3' enhancer) sequences and a remote locus control region (LCR), in part, through gene transfer in placental and nonplacental tumor cells. However, low levels of endogenous hCS/GH-V transcripts are reported in the same cells compared with term placenta, suggesting that chromatin structure, or regulatory region accessibility, versus transcription factor availability contributes to the relatively low levels. To assess individual hCS-A, CS-B, and GH-V gene expression in placental and nonplacental tumor cells and the effect of increasing chromatin accessibility by inhibiting DNA methylation and histone deacetylation using 5-aza-2'-deoxycytidine (azadC) and trichostatin A (TSA). Low levels of hCS-A, CS-B, and GH-V were detected in placental and nonplacental tumor cells compared with term placenta. A significant >5-fold increase in activity was seen in placental, but not nonplacental, cells transfected with hybrid hCS promoter luciferase genes containing 3' enhancer sequences. Pretreatment of placental JEG-3 cells with azadC resulted in a >10-fold increase in hCS-A, CS-B, and GH-V RNA levels with TSA treatment compared with TSA treatment alone. This effect was specific as reversing the treatment regimen did not have the same effect. An assessment of hyperacetylated H3/H4 in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure, including the hCS 3' enhancer sequences and LCR. These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3' enhancer sequences to available placental enhancer transcription factors.

Heart-specific expression of FGF-16 and a potential role in postnatal cardioprotection.

Fibroblast growth factor 16 (FGF-16) was originally cloned from rat heart. Subsequent investigation of mouse FGF-16, including generation of null mice, revealed a specific pattern of expression in the endocardium and epicardium, and role for FGF-16 during embryonic heart development. FGF-16 is expressed mainly in brown adipose tissue during rat embryonic development, but is expressed mainly in the murine heart after birth. There is also an apparent switch from limited endocardial and epicardial expression in the embryo to the myocardium in the perinatal period. The FGF-16 gene and its location on the X chromosome are conserved between human and murine species, and no other member of the FGF family shows this pattern of spatial and temporal expression. The human and murine FGF-16 gene promoter regions also share an equivalent location for TATA sequences, as well as adjacent putative binding sites for transcription factors linked to cardiac expression and response to stress. Recent evidence has implicated nonsense mutation of FGF-16 with increased cardiovascular risk, and FGF-16 supplementation with cardioprotection. Here we review the important role of FGF-16 in embryonic heart development, its gene regulation, and evidence for FGF-16 as an endogenous and exogenous cardiac-specific and protective factor in the postnatal heart. Moreover, given the conservation of the FGF-16 gene and its chromosomal location between species, the question of support for a cardiac role in the human population is also considered.

Energy homeostasis targets chromosomal reconfiguration of the human GH1 locus.

Levels of pituitary growth hormone (GH), a metabolic homeostatic factor with strong lipolytic activity, are decreased in obese individuals. GH declines prior to the onset of weight gain in response to excess caloric intake and hyperinsulinemia; however, the mechanism by which GH is reduced is not clear. We used transgenic mice expressing the human GH (hGH) gene, GH1, to assess the effect of high caloric intake on expression as well as the local chromosome structure of the intact GH1 locus. Animals exposed to 3 days of high caloric intake exhibited hyperinsulinemia without hyperglycemia and a decrease in both hGH synthesis and secretion, but no difference in endogenous production of murine GH. Efficient GH1 expression requires a long-range intrachromosomal interaction between remote enhancer sequences and the proximal promoter region through "looping" of intervening chromatin. High caloric intake disrupted this interaction and decreased both histone H3/H4 hyperacetylation and RNA polymerase II occupancy at the GH1 promoter. Incorporation of physical activity muted the effects of excess caloric intake on insulin levels, GH1 promoter hyperacetylation, chromosomal architecture, and expression. These results indicate that energy homeostasis alters postnatal hGH synthesis through dynamic changes in the 3-dimensional chromatin structure of the GH1 locus, including structures required for cell type specificity during development.

Identification of three novel FGF16 mutations in X-linked recessive fusion of the fourth and fifth metacarpals and possible correlation with heart disease.

Nonsense mutations in FGF16 have recently been linked to X-linked recessive hand malformations with fusion between the fourth and the fifth metacarpals and hypoplasia of the fifth digit (MF4; MIM#309630). The purpose of this study was to perform careful clinical phenotyping and to define molecular mechanisms behind X-linked recessive MF4 in three unrelated families. We performed whole-exome sequencing, and identified three novel mutations in FGF16. The functional impact of FGF16 loss was further studied using morpholino-based suppression of fgf16 in zebrafish. In addition, clinical investigations revealed reduced penetrance and variable expressivity of the MF4 phenotype. Cardiac disorders, including myocardial infarction and atrial fibrillation followed the X-linked FGF16 mutated trait in one large family. Our findings establish that a mutation in exon 1, 2 or 3 of FGF16 results in X-linked recessive MF4 and expand the phenotypic spectrum of FGF16 mutations to include a possible correlation with heart disease.