Felino R. Cagampang

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.

Maternal high-fat feeding primes steatohepatitis in adult mice offspring, involving mitochondrial dysfunction and altered lipogenesis gene expression.

Nonalcoholic fatty liver disease (NAFLD) describes an increasingly prevalent spectrum of liver disorders associated with obesity and metabolic syndrome. It is uncertain why steatosis occurs in some individuals, whereas nonalcoholic steatohepatitis (NASH) occurs in others. We have generated a novel mouse model to test our hypothesis: that maternal fat intake contributes to the development of NAFLD in adult offspring. Female mice were fed either a high‐fat (HF) or control chow (C) diet before and during gestation and lactation. Resulting offspring were fed either a C or a HF diet after weaning, to generate four offspring groups; HF/HF, HF/C, C/HF, C/C. At 15 weeks of age, liver histology was normal in both the C/C and HF/C offspring. Kleiner scoring showed that although the C/HF offspring developed nonalcoholic fatty liver, the HF/HF offspring developed NASH. At 30 weeks, histological analysis and Kleiner scoring showed that both the HF/C and C/HF groups had NAFLD, whereas the HF/HF had a more severe form of NASH. Therefore, exposure to a HF diet in utero and during lactation contributes toward NAFLD progression. We investigated the mechanisms by which this developmental priming is mediated. At 15 weeks of age, hepatic mitochondrial electron transport chain (ETC) enzyme complex activity (I, II/III, and IV) was reduced in both groups of offspring from HF‐fed mothers (HF/C and HF/HF). In addition, measurement of hepatic gene expression indicated that lipogenesis, oxidative stress, and inflammatory pathways were up‐regulated in the 15‐week‐old HF/C and HF/HF offspring. Conclusion: Maternal fat intake contributes toward the NAFLD progression in adult offspring, which is mediated through impaired hepatic mitochondrial metabolism and up‐regulated hepatic lipogenesis. (HEPATOLOGY 2009.)

Long-term maternal high-fat feeding from weaning through pregnancy and lactation predisposes offspring to hypertension, raised plasma lipids and fatty liver in mice

In rodents, adverse prenatal nutrition, such as a maternal diet rich in fat during pregnancy, enhances susceptibility of the offspring to hypertension, type 2 diabetes and other features of the human metabolic syndrome in adulthood. However, previous experimental studies were confined to short-term modifications of the maternal diet during pregnancy and/or lactation periods, a situation uncommon in humans. Moreover in humans, the offspring may also consume a high-fat diet, which may take them beyond the range to which their development has adapted them to respond healthily. We examined in C57 mice the effects on offspring of feeding their mothers a high-fat (HF) or standard chow (C) diet from weaning through pregnancy and lactation, and whether there are additive phenotypic effects of feeding the offspring an HF diet from weaning to adulthood (dam-offspring dietary group HF-HF). This group was compared with offspring from HF-fed dams fed a C diet from weaning to adulthood (HF-C) and offspring from C-fed mothers fed the C or HF diet (C-C and HF-C, respectively). HF-HF, HF-C and C-HF adult female offspring were heavier, fatter, and had raised serum cholesterol and blood pressure compared with C-C female offspring. We observed a similar trend in male offspring except for the HF-C group which was not heavier or fatter than male C-C offspring. Histology showed lipid vacuoles within hepatocytes in the HF-HF, HF-C and C-HF but not the CC offspring. Serum C-reactive protein was elevated in female (C-HF and HF-HF) but not in male offspring. Elevated blood pressure in the HF-C and C-HF groups was attenuated in the HF-HF group in males but not in females. These findings indicate that long-term consumption of an HF diet by the mother predisposes her offspring to developing a metabolic syndrome-like phenotype in adult life, although cardiovascular effects of an HF diet are related to sex specificity in the HF-HF group.

Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring.

BackgroundmiRNAs play important roles in the regulation of gene functions. Maternal dietary modifications during pregnancy and gestation have long-term effects on the offspring, but it is not known whether a maternal high fat (HF) diet during pregnancy and lactation alters expression of key miRNAs in the offspring.ResultsWe studied the effects of maternal HF diet on the adult offspring by feeding mice with either a HF or a chow diet prior to conception, during pregnancy and lactation, and all offspring were weaned onto the same chow diet until adulthood. Maternal HF fed offspring had markedly increased hepatic mRNA levels of peroxisome proliferator activated receptor-alpha (ppar-alpha) and carnitine palmitoyl transferase-1a (cpt-1a) as well as insulin like growth factor-2 (Igf2). A HF diet induced up-regulation of ppar-alpha and cpt-1a expression in the wild type but not in Igf2 knock out mice. Furthermore, hepatic expression of let-7c was also reduced in maternal HF fed offspring. Among 579 miRNAs measured with microarray, ~23 miRNA levels were reduced by ~1.5-4.9-fold. Reduced expression of miR-709 (a highly expressed miRNA), miR-122, miR-192, miR-194, miR-26a, let-7a, let7b and let-7c, miR-494 and miR-483* (reduced by ~4.9 fold) was validated by qPCR. We found that methyl-CpG binding protein 2 was the common predicted target for miR-709, miR-let7s, miR-122, miR-194 and miR-26a using our own purpose-built computer program.ConclusionMaternal HF feeding during pregnancy and lactation induced co-ordinated and long-lasting changes in expression of Igf2, fat metabolic genes and several important miRNAs in the offspring.

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease‐related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF‐1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety‐related behaviour in open field activities in male and female offspring (P < 0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P < 0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P < 0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P < 0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P < 0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post‐fertilization preimplantation development which may reflect the relative contribution of the paternal genome.

Meeting Report on the 3rd International Congress on Developmental Origins of Health and Disease (DOHaD)

Developmental origins of health and disease (DOHaD) focuses on the earliest stages of human development, and provides a novel paradigm to complement other strategies for lifelong prevention of common chronic health conditions. The 3rd International Congress on DOHaD, held in 2005, retained the most popular features from the first two biannual Congresses, while adding a number of innovations, including increased emphasis on implications of DOHaD for the developing world; programs for trainees and young investigators; and new perspectives, including developmental plasticity, influences of social hierarchies, effects of prematurity, and populations in transition. Emerging areas of science included, first, the controversial role of infant weight gain in predicting adult obesity, diabetes, and cardiovascular disease. Second, in the era of epidemic obesity, paying attention to the over-nourished fetus is as important as investigating the growth retarded one. Third, environmental toxins appear to have abroad range of long-lasting effects on the developing human. Fourth, epigenetic mechanisms could unite several strands of human and animal observations, and explain how genetically identical individuals raised in similar postnatal environments can nonetheless develop widely differing phenotypes. Improving the environment to which an individual is exposed during development may be as important as any other public health effort to enhance population health world wide.

Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the Syrian hamster

In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks.

Maternal Periconceptional and Gestational Low Protein Diet Affects Mouse Offspring Growth, Cardiovascular and Adipose Phenotype at 1 Year of Age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P<0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health.

Statin Treatment in Hypercholesterolemic Pregnant Mice Reduces Cardiovascular Risk Factors in Their Offspring

Increasing evidence suggests that hypercholesterolemia during pregnancy initiates pathogenic events in the fetus leading to increased risk of cardiovascular disease in the adult offspring. In this study we examined in mice whether pharmacological intervention using statins in late pregnancy could alleviate the detrimental effects of a high-fat, high-cholesterol (45% fat) maternal diet on the health of the dams and their offspring. Pregnant C57 mice on high-fat, high-cholesterol diet were given the 3hydroxy3methylglutaryl-coenzyme A reductase inhibitor pravastatin in the drinking water (5 mg/kg of body weight per day) in the second half of pregnancy and during lactation to lower cholesterol and improve postweaning maternal blood pressure. Weaned offspring were then fed the high-fat, high-cholesterol diet until adulthood (generating dam/offspring dietary groups high-fat, high-cholesterol/high-fat, high-cholesterol and high-fat, high-cholesterol plus pravastatin during the second half of pregnancy and lactation/high-fat, high-cholesterol). These groups were compared with offspring from mothers fed standard chow (control), which were then fed control diet to adulthood (control/control). Compared with high-fat, high-cholesterol, high-fat, high-cholesterol plus pravastatin during second half of pregnancy and lactation dams showed significantly reduced total cholesterol concentrations and reduced systolic blood pressure. The high-fat, high-cholesterol plus pravastatin during second half of pregnancy and lactation/high-fat, high-cholesterol offspring were significantly lighter, less hypertensive, and more active compared with the high-fat, high-cholesterol/high-fat, high-cholesterol group. Total serum and low-density lipoprotein cholesterol concentrations were significantly lower, and high-density lipoprotein cholesterol concentrations were raised in high-fat, high-cholesterol plus pravastatin during the second half of pregnancy and lactation/high-fat, high-cholesterol offspring, compared with the high-fat, high-cholesterol/high-fat, high-cholesterol group. The control/control offspring showed the lowest blood pressure and cholesterol levels. These findings indicate that the cholesterol-lowering effect of statins in pregnant dams consuming a high-fat, high-cholesterol diet leads to reduced cardiovascular risk factors in offspring that are sustained into adulthood.

Evidence for an endogenous per1- and ICER-independent seasonal timer in the hamster pituitary gland

Most mammals use changing annual day‐length cycles to regulate pineal melatonin secretion and thereby drive many physiological rhythms including reproduction, metabolism, immune function, and pelage. Prolonged exposure to short winter day lengths results in refractoriness, a spontaneous reversion to long‐day physiological status. Despite its critical role in the timing of seasonal rhythms, refractoriness remains poorly understood. The aim of this study was therefore to describe cellular and molecular mechanisms driving the seasonal secretion of a key hormone, prolactin, in refractory Syrian hamsters. We used recently developed single cell hybridization and reporter assays to show that this process is initiated by timed reactivation of endocrine signaling from the pars tuberalis (PT) region of the pituitary gland, a well‐defined melatonin target site, causing renewed activation of prolactin gene expression. This timed signaling is independent of per1 clock gene expression in the suprachiasmatic nuclei and PT and of melatonin secretion, which continue to track day length. Within the PT, there is also a continued short day‐like profile of ICER expression, suggesting that the change in hormone secretion is independent of cAMP signaling. Our data thus identify the PT as a key anatomical structure involved in endogenous seasonal timing mechanisms, which breaks from prevailing day length‐induced gene expression.—Johnston, J. D., Cagampang, F. R. A., Stirland, J. A., Carr, A.‐J. F., White, M. R. H., Davis, J. R. E., Loudon, A. S. I. FASEB J. 17, 810–815 (2003)