Unique Genetic and Histological Signatures of Mouse Pericardial Adipose Tissue.

Obesity is a major risk factor for a plethora of metabolic disturbances including diabetes and cardiovascular disease. Accumulating evidence is showing that there is an adipose tissue depot-dependent relationship with obesity-induced metabolic dysfunction. While some adipose depots, such as subcutaneous fat, are generally metabolically innocuous, others such as visceral fat, are directly deleterious. A lesser known visceral adipose depot is the pericardial adipose tissue depot. We therefore set out to examine its transcriptional and morphological signature under chow and high-fat fed conditions, in comparison with other adipose depots, using a mouse model. Our results revealed that under chow conditions pericardial adipose tissue has uncoupling-protein 1 gene expression levels which are significantly higher than classical subcutaneous and visceral adipose depots. We also observed that under high-fat diet conditions, the pericardial adipose depot exhibits greatly upregulated transcript levels of inflammatory cytokines. Our results collectively indicate, for the first time, that the pericardial adipose tissue possesses a unique transcriptional and histological signature which has features of both a beige (brown fat-like) but also pro-inflammatory depot, such as visceral fat. This unique profile may be involved in metabolic dysfunction associated with obesity.

Defective NOTCH signaling drives increased vascular smooth muscle cell apoptosis and contractile differentiation in bicuspid aortic valve aortopathy: A review of the evidence and future directions.

Bicuspid aortic valve (BAV) disease remains the most common congenital cardiac disease and is associated with an increased risk of potentially fatal aortopathy including aortic aneurysm and dissection. Mutations in the NOTCH1 gene are one of only a few genetic anomalies identified in BAV disease; however evidence for defective NOTCH signaling, and its involvement in the characteristic histological changes of VSMC apoptosis and differentiation in ascending aortae of BAV patients is lacking. This review scrutinizes the evidence for the interactions of NOTCH signaling, cellular differentiation and apoptosis in the context of aortic VSMCs and provides focus for future research efforts in the diagnosis of BAV aortopathy and prevention of catastrophic complications through NOTCH signaling manipulation.

The influence of a high fat diet on bone and soft tissue formation in Matrix Gla Protein knockout mice.

Studies suggest bone growth and development are influenced by maternal nutrition, during intrauterine and early postnatal life. This study assessed the role of MGP and a maternal high fat diet on vitamin K-dependent proteins' gene expression and their impact on bone formation. Knockout (KO) offspring were smaller than wild type (WT) littermates, yet possessed the same volume of intrascapular brown adipose tissue. The total proportion of body fat was reduced, but only in animals on a control diet. Lung air volume was observed to be comparable in both KO and WT animals on the same diet. The degree of aortic calcification was reduced in KO animals maintained on a HF diet. KO females on the high fat diet showed reduced cortical bone volume and thickness in the femur and tibia. Gene expression levels of GGCX and VKOR were reduced in control fed KO animals suggesting a potential link between gene expression levels of MGP, GGCX, and VKOR and total volumes of bone, calcified soft tissue, and iBAT; with implications for modulation of body length and mass. Our results confirm the important role for vitamin K in bone and calcified soft tissue, but now extend this role to include iBAT.

MNK1 and MNK2 mediate adverse effects of high-fat feeding in distinct ways.

The MAP kinase-interacting kinases (MNK1 and MNK2) are non-essential enzymes which are activated by MAP kinases. They are implicated in controlling protein synthesis. Here we show that mice in which the expression of either MNK1 or MNK2 has been knocked out (KO) are protected against adverse effects of high-fat feeding, and in distinct ways. High-fat diet (HFD)-fed MNK2-KO show less weight gain than wild-type animals, and improved glucose tolerance, better insulin sensitivity and markedly diminished adipose tissue inflammation. This suggests MNK2 plays a role in adipogenesis and/or lipogenesis and in macrophage biology. MNK1-KO/HFD mice show better glucose tolerance and insulin sensitivity, but gain weight and show similar adipose inflammation to WT animals. These data suggest MNK1 participates in mediating HFD-induced insulin resistance. Our findings reveal distinct roles for the MNKs in a novel area of disease biology, metabolic dysfunction, and suggests they are potential new targets for managing metabolic disease.

Maternal high-fat diet and offspring expression levels of vitamin K-dependent proteins.

Studies suggest that bone growth and development and susceptibility to vascular disease in later life are influenced by maternal nutrition during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including osteocalcin, matrix Gla protein, periostin, and growth-arrest specific- protein 6, in both bone and vascular development. We have examined whether there are alterations in these VKDPs in bone and vascular tissue from offspring of mothers subjected to a nutritional challenge: a high-fat diet during pregnancy and postnatally, using 6-week-old mouse offspring. Bone site-specific and sex-specific differences across femoral and vertebral bone in male and female offspring were observed. Overall a high-fat maternal diet and offspring diet exacerbated the bone changes observed. Sex-specific differences and tissue-specific differences were observed in VKDP levels in aorta tissue from high-fat diet-fed female offspring from high-fat diet-fed mothers displaying increased levels of Gas6 and Ggcx compared with those of female controls. In contrast, differences were seen in VKDP levels in femoral bone of female offspring with lower expression levels of Mgp in offspring of mothers fed a high-fat diet compared with those of controls. We observed a significant correlation in Mgp expression levels within the femur to measures of bone structure of the femur and vertebra, particularly in the male offspring cohort. In summary, the current study has highlighted the importance of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

Long-term statin administration to dams on high-fat diet protects not only them but also their offspring from cardiovascular risk.

We aimed to study the effects of long-term statin administration to high fat (HF)-fed female mice from the time they were weaned through to pregnancy and lactation on cardiovascular and metabolic risk factors in their HF-fed offspring. Female C57 mice on HF (45% kcal fat) were given pravastatin in their drinking water from the time they were weaned, during pregnancy and lactation. Weaned offspring were then fed an HF diet until adulthood generating the dam/offspring dietary groups HF/HF and HF plus pravastatin from the time dams were weaned, during pregnancy and lactation/HF (HF+S/HF). These groups were compared with offspring from mothers fed standard chow (control) which were then fed a control diet up to adulthood (control/control; C/C). HF+S dams showed significantly reduced total cholesterol concentrations and systolic blood pressure (SBP) versus HF dams. The reduction in total cholesterol and SBP in the HF+S dams did not restore values to those observed in the C group. Both male and female HF+S/HF offspring were significantly lighter in weight, and had lower SBP and serum cholesterol concentrations versus HF/HF. HF/HF offspring had elevated C-reactive protein levels but HF+S/HF animals of both sexes had reduced levels similar to those found in the C/C group. Long-term pravastatin administration to dams not only protects them from the deleterious effects of an HF diet, but this long-term maternal statin protection also has an equal and permanent effect in both male and female offspring up to their adult life, which is a novel finding.

Developmental exposure to bisphenol A leads to cardiometabolic dysfunction in adult mouse offspring.

Bisphenol A (BPA) is a chemical compound that has adverse health outcomes in adults when exposed during the perinatal period. However, its effect on cardiovascular function remains to be elucidated. In this study, we examined the effects of daily administration of BPA to pregnant mice from gestational days 11 to 19 on cardiometabolic outcomes in the adult offspring. Prenatal BPA exposure resulted in altered growth trajectory and organ size, increase adiposity and impaired glucose homeostasis in male and female offspring. In addition, these BPA offspring exhibited raised systolic blood pressure, and in the males this was accompanied by impaired vascular tone. The aortas in females, but not in males, from the BPA group also showed reduced estrogen receptor gene expression. These results indicate that prenatal exposure to BPA increased susceptibility of the offspring to developing cardiovascular and metabolic dysfunction later in life.

Variation in stability of housekeeping genes in endometrium of healthy and polycystic ovarian syndrome women.

BACKGROUND: The use of housekeeping genes (HKG) as internal controls for real-time qPCR studies of gene expression is based on the assumption of their inherent stability. However, it is unclear whether this stability is maintained in disease states. In order to test this, the present study investigated the expression of specific HKG in the endometrium of healthy and polycystic ovarian syndrome (PCOS) women. METHODS: Endometrial tissue samples were taken from women with PCOS (n= 9) and controls (n= 10). The stability of nine candidate reference genes in the endometrial tissues were evaluated; four encode mitochondrial proteins [ATP5B, succinate dehydrogenase complex subunit A (SDHA), cytochrome c-1, glyceraldehyde-3-phosphatedehydrogenase], two encode ribosomal protein genes (18s ribosomal RNA, ribosomal protein L13A), one for cell structure (SDHA), one for cell signalling (beta actin, ACTB) and one involved in DNA repair (topoisomerase I, TOP1). The expression stability of these HKGs was calculated using geNORM qbasePLUS software, with stability defined by M-values, where higher M-value indicating less stability. In addition, changes in their cycle threshold values were analysed to determine direction of change between groups, and a Mann-Whitney U-test was used to determine statistical differences in expression. RESULTS: The most stable HKGs observed across both PCOS endometrium were found to be YWHAZ, CYC1 and ACTB. Further TOP1 demonstrated higher gene expression in the endometrium from PCOS women compared with those from healthy women. CONCLUSIONS: Of the nine HKGs examined, only YWHAZ, CYC1 and ACTB were stable in both control and PCOS endometrium: these should therefore be used as internal controls for quantitative reverse transcription-polymerase chain reaction analysis. Published discrepancies between endometrial gene expression studies may therefore be due in part to in the inappropriate HKG selection, and future gene expression studies should be based on HKG of known stability in both the disease and healthy states to avoid erroneous interpretation of results.

Effect of a low-protein diet during pregnancy on expression of genes involved in cardiac hypertrophy in fetal and adult mouse offspring.

Gene markers for cardiomyocyte growth, proliferation and remodeling were examined in mouse fetuses and adult male offspring exposed to maternal low-protein (LP) diet during pregnancy. Whole heart volume, measured by magnetic resonance imaging, was smaller in day 15 LP fetuses v. those from chow-fed dams (C), whereas heart volume was greater in adult LP v. C offspring. These LP offspring were hypertensive and had larger cardiomyocytes v. C animals. The mRNA levels of cyclin G1, a marker for cell growth, were lower in LP fetal hearts v. C hearts, but similar in the left ventricle of adult LP and C offspring. Opposite trends were found in brain natriuretic peptide levels (a marker of cardiac hypertrophy). Thus, maternal LP during pregnancy results in smaller fetal hearts and is accompanied by changes in expression of genes involved in cardiomyocyte growth, which are associated with cardiac hypertrophy and hypertension in adulthood.

Maternal high-fat diet: effects on offspring bone structure.

UNLABELLED: Peak bone mass is believed to partly be programmed in utero. Mouse dams and offspring were given a high-fat diet and offspring studied as adults. Female offspring from high-fat dams exhibited altered trabecular structure indicative of in utero programming. In utero nutrition has consequences in later life. INTRODUCTION: Epidemiological studies suggest that skeletal growth is programmed during intrauterine and early postnatal life. We hypothesise that development of optimal peak bone mass has, in part, a foetal origin and investigated this using a mouse model of maternal dietary fat excess. METHODS: Offspring from mouse dams fed either standard chow (C) or lifetime high-fat diet (HF) were maintained on a HF diet to adulthood. Femur samples were taken at 30 weeks of age and bone structure, adiposity and strength analysed. Sample sizes were four to six for each sex and each diet group. RESULTS: Offspring from HF-fed dams showed increased adiposity in the femur in comparison to offspring from C-fed dams. Female offspring from HF dams exhibited altered trabecular structure indicative of in utero programming. CONCLUSIONS: A maternal HF diet during pregnancy increases bone marrow adiposity and alters bone structure in their offspring.

Skeletal bone morphology is resistant to the high amplitude seasonal leptin cycle in the Siberian hamster.

Recent studies have suggested that the adipocyte-derived hormone, leptin, plays a role in the regulation of metabolism. Here, we tested this hypothesis in the seasonally breeding Siberian hamster, as this species exhibits profound seasonal changes in adiposity and circulating leptin concentrations driven by the annual photoperiodic cycle. Male hamsters were kept in either long (LD) or short (SD) photoperiods. Following exposure to short photoperiods for 8 weeks animals exhibited a significant weight-loss and a 16-fold reduction of serum leptin concentrations. At Week 9, animals in both photoperiods were infused with leptin or PBS via osmotic mini-pump for 14 days. Chronic leptin infusion mimicked LD-like concentrations in SD-housed animals and caused a further decline in body weight and adipose tissue. In LD-housed animals, leptin infusion resulted in a significant elevation of serum concentrations above natural LD-like levels, but had no discernable effect on body weight or overall adiposity. Both bending and compression characteristics and histomorphometric measurements of trabecular bone mass were unaltered by leptin treatment or photoperiod. Our data therefore show that despite a high natural amplitude cycle of leptin, this hormone has no apparent role in the regulation of bone metabolism, and therefore do not support recent propositions that this hormone is an important component in the metabolism of bone tissue.

The tau mutation in the Syrian hamster differentially reprograms the circadian clock in the SCN and peripheral tissues.

The hypothalamic suprachiasmatic nuclei (SCN), the principal circadian oscillator in mammals, are synchronized to the solar day by the light-dark cycle, and in turn, they coordinate circadian oscillations in peripheral tissues. The tau mutation in the Syrian hamster is caused by a point mutation leading to a deficiency in the ability of Casein Kinase 1epsilon to phosphorylate its targets, including circadian PER proteins. How this accelerates circadian period in neural tissues is not known, nor is its impact on peripheral circadian oscillators established. We show that this mutation has no effect on per mRNA expression nor the nuclear accumulation of PER proteins in the SCN. It does, however, accelerate the clearance of PER proteins from the nucleus to an extent sufficient to explain the shortened circadian period of behavioral rhythms. The mutation also has novel, unanticipated consequences for circadian timing in the periphery, including tissue-specific phase advances and/or reduced amplitude of circadian gene expression. The results suggest that the tau mutation accelerates a specific phase, during mid-late subjective night of the SCN circadian feedback loop, rather than cause a global compression of the entire cycle. This reprogrammed output from the clock is associated with peripheral desynchrony, which in turn could account for impaired growth and metabolic efficiency of the mutant.

Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalis-derived factor.

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8-12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.

Posttranslational mechanisms regulate the mammalian circadian clock.

We have examined posttranslational regulation of clock proteins in mouse liver in vivo. The mouse PERIOD proteins (mPER1 and mPER2), CLOCK, and BMAL1 undergo robust circadian changes in phosphorylation. These proteins, the cryptochromes (mCRY1 and mCRY2), and casein kinase I epsilon (CKIepsilon) form multimeric complexes that are bound to DNA during negative transcriptional feedback. CLOCK:BMAL1 heterodimers remain bound to DNA over the circadian cycle. The temporal increase in mPER abundance controls the negative feedback interactions. Analysis of clock proteins in mCRY-deficient mice shows that the mCRYs are necessary for stabilizing phosphorylated mPER2 and for the nuclear accumulation of mPER1, mPER2, and CKIepsilon. We also provide in vivo evidence that casein kinase I delta is a second clock relevant kinase.

Leptin acts on metabolism in a photoperiod-dependent manner, but has no effect on reproductive function in the seasonally breeding Siberian hamster (Phodopus sungorus).

Leptin may play a role in appetite regulation and metabolism, but its reproductive role is less clear. In photoperiodic Siberian hamsters, seasonal changes in fatness, leptin gene expression, and metabolism occur synchronously with activation or suppression of reproduction, analogous to puberty. Here, we test the hypothesis that seasonal changes in leptin secretion mediate the photoperiodic regulation of reproduction. Mature male and ovariectomized estrogen-treated female Siberian hamsters were kept in long (LD; 16 h of light, 8 h of darkness) or short days (SD; 8 h of light, 16 h of darkness) for 8 weeks, and recombinant murine leptin (15 microg/day) was infused for 2 weeks via osmotic minipumps. SD hamsters exhibited significant weight and fat losses, reduced serum leptin and food intake, and suppressed pituitary LH concentration. Leptin did not suppress food intake over the 2-week treatment on either photoperiod, but significantly reduced fat reserves in SD hamsters. Leptin had no significant effect on pituitary LH concentrations in either sex or photoperiod or on testicular size and testosterone concentrations in males. These results suggest hamsters are more responsive to leptin on SD than on LD and that effects on food intake and fat loss can be dissociated in this species. Our data suggest that leptin does not mediate photoperiodic reproductive changes.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans.

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERalpha-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.

Circadian regulation of prion protein messenger RNA in the rat forebrain: a widespread and synchronous rhythm.

Although the expression of the normal prion protein in the host is critical to the development of transmissible spongiform encephalopathies, the physiological role of this protein and the processes regulating its expression remain obscure. We now report that the messenger RNA for the prion protein is regulated in the rat brain in a marked circadian manner not only in the suprachiasmatic nuclei, the principal site for the generation of mammalian circadian rhythms, but also in other forebrain regions. The data show a remarkable consistency in the concurrence of a single peak of prion protein messenger RNA at each of the sites early in the animal's phase of increased locomotor activity; behavioural arousal does not, however, appear to affect this expression. We believe this to be the first study demonstrating that the expression of prion protein messenger RNA can change over a relatively short period in vivo. The results are discussed with reference to the range of recently discovered "clock-related" transcripts which also have widespread tissue expression; these include the messenger RNAs for D-box binding protein and thyroid embryonic factor, transcription factors which bind to the prion protein promoter.

Adrenomedullectomy prevents the suppression of pulsatile luteinising hormone release during fasting in female rats.

Fasting inhibits the pulsatile secretion of luteinising hormone (LH) in female rats, an effect which is potentiated by the presence of oestradiol (E2). We have previously described various pharmacological or surgical treatments that can rapidly restore the pulses in a fasting animal. Nevertheless, the central and peripheral mechanisms that mediate this suppression of the pulses remain unclear. We have recently shown that adrenomedullectomy prevents the suppression of LH pulses by insulin-induced hypoglycaemia, a state which activates the sympathoadrenal axis. The present study was undertaken to establish whether this axis might contribute to the loss of the pulses that occurs in ovariectomised E2-treated rats that have been fasted for 48 h. Following sham adrenomedullectomy LH pulses were observed in animals fed ad libitum; after 48 h of fasting the animals that had received this sham procedure showed a significant suppression of LH levels and LH pulse frequency. In contrast, adrenomedullectomy prevented the inhibition of the pulses by 48 h of fasting; it had no effect on the pulses in the absence of fasting. These results suggest that adrenomedullary activity plays a significant role in the fasting-induced suppression of LH pulses in rats.

Circadian changes in PACAP type 1 (PAC1) receptor mRNA in the rat suprachiasmatic and supraoptic nuclei.

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP), denoted as PAC1, is expressed in the suprachiasmatic nuclei (SCN). Since the circadian clock demonstrates phase-dependent sensitivity to PACAP, we have used in situ hybridization histochemistry to examine whether PAC1 mRNA is differentially expressed in the rat SCN across the 24-h cycle. There was a significant variation in PAC1 mRNA within the SCN and supraoptic nuclei during the light-dark cycle and in constant darkness, with peaks at the middle of both the real and subjective day and night; no significant variation was observed in the cingulate cortex. The results suggest that the phase-dependent actions of PACAP on the clock may involve phase-specific changes in the availability of PAC1 receptors within the SCN.

Circadian changes of type II adenylyl cyclase mRNA in the rat suprachiasmatic nuclei.

Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme which, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization we have found a biphasic variation in AC-II mRNA within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex showed no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN.