Rapidly alternating photoperiods disrupt central and peripheral rhythmicity and decrease plasma glucose, but do not affect glucose tolerance or insulin secretion in sheep.

Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. To determine whether these effects occur in a large diurnal animal, we assessed the impact of circadian rhythm disruption upon metabolic function in sheep. Adult ewes (n = 7) underwent 3 weeks of a control 12 h light-12 h dark photoperiod, followed by 4 weeks of rapidly alternating photoperiods (RAPs) whereby the time of light exposure was reversed twice each week. Measures of central (melatonin secretion and core body temperature) and peripheral rhythmicity (clock and metabolic gene expression in skeletal muscle) were obtained over 24 h in both conditions. Metabolic homeostasis was assessed by glucose tolerance tests and 24 h glucose and insulin profiles. Melatonin and core body temperature rhythms resynchronized within 2 days of the last photoperiod shift. High-amplitude Bmal1, Clock, Nr1d1, Cry2 and Per3 mRNA rhythms were apparent in skeletal muscle, which were phase advanced by up to 3.5 h at 2 days after the last phase shift, whereas Per1 expression was downregulated at this time. Pparα, Pgc1α and Nampt mRNA were constitutively expressed in both conditions. Nocturnal glucose concentrations were reduced following chronic phase shifts (zeitgeber time 0, -5.5%; zeitgeber time 12, -2.9%; and zeitgeber time 16, -5.7%), whereas plasma insulin, glucose tolerance and glucose-stimulated insulin secretion were not altered. These results demonstrate that clock gene expression within ovine skeletal muscle oscillates over 24 h and responds to changing photoperiods. However, metabolic genes which link circadian and metabolic clocks in rodents were arrhythmic in sheep. Differences may be due to the ruminant versus monogastric digestive organization in each species. Together, these results demonstrate that despite disruptions to central and peripheral rhythmicity following exposure to rapidly alternating photoperiods, there was minimal impact on glucose homeostasis in the sheep.

Maternal obesity or weight loss around conception impacts hepatic fatty acid metabolism in the offspring.

OBJECTIVE: To determine the impact of maternal obesity or weight loss during the periconceptional period on programming of lipid metabolism in the liver of the offspring. METHODS: An embryo transfer model was used to investigate the effects of exposure to either maternal obesity and/or weight loss before and for 1-week post-conception on the abundance of key molecules regulating hepatic fatty acid oxidation and lipid synthesis in the 4-month-old lamb. RESULTS: Periconceptional maternal obesity resulted in decreased hepatic PPARα, PGC1α and GCN5 abundance and increased hepatic SIRT1 and AMPKα1, AMPKα2 and SREBP1 abundance in the offspring. Maternal weight loss in obese ewes did not ablate all of these effects of maternal obesity on hepatic metabolism in the lamb. Weight loss in normal weight ewes also resulted in decreased hepatic PGC1α and GCN5 and increased AMPKα2 abundance in the offspring. CONCLUSIONS: Exposure of the oocyte/embryo to either maternal obesity or weight loss during the periconceptional period has long term consequences for hepatic lipid metabolism. These findings highlight the sensitivity of the early embryo to maternal nutrition and the need for dietary interventions which maximize metabolic benefits and minimize metabolic costs for the next generation.

Metabolic consequences of timed feeding in mice.

The time of day at which meals are consumed is known to impact on behaviour as well as physiological systems. In this study we investigated the behavioural and physiological effects of restricting access to food to the light or dark period in mice maintained on either long or short photoperiods. In both photoperiods, wheel running commenced upon the onset of darkness and was generally confined to the period of darkness. Provision of food during light provoked an anticipatory burst of activity several hours before feeding in both photoperiods. After 28 days on the feeding schedule, body weight was unaffected by either photoperiod or feeding time. Plasma insulin was increased and glucose and triglycerides tended to be lower in mice fed during the light period and sampled 2 h after lights off compared to the dark fed mice. Mice fed during the light while on long day length had improved glucose tolerance and whole body insulin tolerance when tested 2 h after lights on. This was not evident in mice kept on the short photoperiod. Because these observations were confounded by the time since their last meal, we undertook a study of glucose tolerance across 24 h in mice on the long photoperiod after a 2 hour food withdrawal. A clear rhythm of glucose tolerance was observed in mice fed during the light period with maximal glucose tolerance just prior to the expected presentation of food and minimal tolerance 2 h before lights off. By contrast, no rhythm in glucose tolerance was observed in the dark fed mice, but maximal glucose tolerance occurred 2 h before lights off. To investigate the evolution of the physiological adaptations, mice on this feeding/photoperiod regime were studied after 7 or 35 days. After 7 days the corticosterone rhythm was not different between light and dark fed mice, but by 35 days peak corticosterone secretion occurred a few hours before food presentation in both groups representing an 8 hour shift. The rhythm of expression of liver Bmal1 mRNA was similar in light and dark fed mice after 7 and 35 days on the schedule while the Per1, Per2, Nr1d1 and Dbp mRNA rhythms were delayed on average by 3.5±1.1 h and 3.7±0.9 h in light fed mice after 7 and 35 days respectively compared to dark fed mice. Rhythms of metabolically important genes were shifted in light fed mice compared to dark fed, by 5 h or became arrhythmic. This study shows that not only circadian rhythms facilitate metabolic control, but also different environmental events, including season and feeding opportunities, alter aspects of circadian and metabolic physiology.

Impact of maternal overnutrition on gluconeogenic factors and methylation of the phosphoenolpyruvate carboxykinase promoter in the fetal and postnatal liver.

BACKGROUND: Exposure to maternal obesity or hyperglycemia increases the risk of obesity and poor glucose tolerance in the offspring. We hypothesized that maternal overnutrition in late pregnancy would result in (i) lower methylation in the promoter region of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C; PCK1) and (ii) higher expression of hepatic gluconeogenic factors in the fetal and postnatal lamb. METHODS: Ewes were fed 100% (n = 18) or ~155% (n = 17) of energy requirements from 115 d gestation, and livers were collected at ~140 d gestation or 30 d postnatal age. RESULTS: Maternal overnutrition resulted in a decrease in hepatic expression of the mitochondrial form of PEPCK (PEPCK-M; PCK2) but not of PEPCK-C or glucose-6-phosphatase (G6PHOS) before and after birth. Hepatic expression of peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), peroxisome proliferator-activated receptor α (PPARα), PEPCK-C, G6PHOS, and 11ß hydroxysteroid dehydrogenase type 1 (11ßHSD1), but not PEPCK-M, was higher in the postnatal lamb compared with that in the fetal lamb. The level of PCK1 methylation was paradoxically approximately twofold higher in the postnatal liver compared with that in the fetal liver. CONCLUSION: Maternal overnutrition programs a decrease in hepatic PEPCK-M in the offspring and as ~50% of total hepatic PEPCK is PEPCK-M, the longer-term consequences of this decrease may be significant.

Maternal dietary restriction during the periconceptional period in normal-weight or obese ewes results in adrenocortical hypertrophy, an up-regulation of the JAK/STAT and down-regulation of the IGF1R signaling pathways in the adrenal of the postnatal lamb.

Maternal dietary restriction during the periconceptional period results in an increase in adrenal growth and in the cortisol stress response in the offspring. The intraadrenal mechanisms that result in the programming of these changes are not clear. Activation of the IGF and the signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways regulate adrenal growth. We have used an embryo transfer model in sheep to investigate the impact of exposure to either dietary restriction in normal or obese mothers or to maternal obesity during the periconceptional period on adrenal growth and function in the offspring. We assessed the adrenal abundance of key signaling molecules in the IGF-I and Janus kinase/STAT/SOCS pathways including IGF-I receptor, IGF-II receptor, Akt, mammalian target of rapamycin, ribosomal protein S6, eukaryotic translation initiation factor 4E-binding protein 1, eukaryotic translation initiation factor 4E, STAT1, STAT3, STAT5, SOCS1, and SOCS3 in female and male postnatal lambs. Maternal dietary restriction in the periconceptional period resulted in the hypertrophy of the adrenocortical cells in the zona fasciculata-reticularis and an up-regulation in STAT1, phospho-STAT1, and phospho-STAT3 (Ser727) abundance and a down-regulation in IGF-I receptor, Akt, and phospho-Akt abundance in the adrenal cortex of the postnatal lamb. These studies highlight that weight loss around the time of conception, independent of the starting maternal body weight, results in the activation of the adrenal Janus kinase/STAT pathway and adrenocortical hypertrophy. Thus, signals of adversity around the time of conception have a long-term impact on the mechanisms that regulate adrenocortical growth.

Dietary restriction in the periconceptional period in normal-weight or obese ewes results in increased abundance of angiotensin-converting enzyme (ACE) and angiotensin type 1 receptor (AT1R) in the absence of changes in ACE or AT1R methylation in the adrenal of the offspring.

Exposure to dietary restriction during the periconceptional period in either normal or obese ewes results in increased adrenal growth and a greater cortisol response to stress in the offspring, but the mechanisms that programme these changes are not fully understood. Activation of the angiotensin type 1 receptor (AT1R) has been demonstrated to stimulate adrenal growth and steroidogenesis. We have used an embryo transfer model in the sheep to investigate the effects of exposure to dietary restriction in normal or obese mothers from before and 1 week after conception on the methylation status, expression, abundance and localisation of key components of the renin-angiotensin system (RAS) in the adrenal of post-natal lambs. Maternal dietary restriction in normal or obese ewes during the periconceptional period resulted in an increase in angiotensin-converting enzyme (ACE) and AT1R abundance in the absence of changes in the methylation status or mRNA expression of ACE and AT1R in the adrenal of the offspring. Exposure to maternal obesity alone also resulted in an increase in adrenal AT1R abundance. There was no effect of maternal dietary restriction or obesity on ACE2 and AT2R or on ERK, calcium/calmodulin-dependent kinase II abundance, and their phosphorylated forms in the lamb adrenal. Thus, weight loss around the time of conception, in both normal-weight and obese ewes, results in changes within the intra-adrenal RAS consistent with increased AT1R activation. These changes within the intra-adrenal RAS system may contribute to the greater adrenal stress response following exposure to signals of adversity in the periconceptional period.

Differential effects of maternal obesity and weight loss in the periconceptional period on the epigenetic regulation of hepatic insulin-signaling pathways in the offspring.

Our aim was to determine the effect of exposure to maternal obesity or to maternal weight loss around conception on the programming of hepatic insulin signaling in the offspring. We used an embryo transfer model in sheep to investigate the effects of exposure to either maternal obesity or to weight loss in normal and obese mothers preceding and for 1 wk after conception on the expression of hepatic insulin-signaling and gluconeogenic factors and key miRNAs involved in insulin signaling in the offspring. We found that exposure to maternal obesity resulted in increased hepatic miR-29b (P<0.05), miR-103 (P<0.01), and miR-107 (P<0.05) expression, a decrease in IR (P<0.05), phopsho-Akt (P<0.01), and phospho-FoxO1 (P<0.01) abundance, and a paradoxical decrease in 11ßHSD1 (P<0.05), PEPCK-C (P<0.01), and PEPCK-M (P<0.05) expression in lambs. These changes were ablated by a period of moderate dietary restriction imposed during the periconceptional period. Maternal dietary restriction alone also resulted in decreased abundance of a separate subset of hepatic insulin-signaling molecules, namely, IRS1 (P<0.05), PDK1 (P<0.01), phospho-PDK1 (P<0.05), and aPKCζ (P<0.05) and in decreased PEPCK-C (P<0.01) and G6Pase (P<0.01) expression in the lamb. Our findings highlight the sensitivity of the epigenome to maternal nutrition around conception and the need for dietary interventions that maximize metabolic benefits and minimize metabolic costs for the next generation.

Characterisation of the maternal response to chronic phase shifts during gestation in the rat: implications for fetal metabolic programming.

Disrupting maternal circadian rhythms through exposure to chronic phase shifts of the photoperiod has lifelong consequences for the metabolic homeostasis of the fetus, such that offspring develop increased adiposity, hyperinsulinaemia and poor glucose and insulin tolerance. In an attempt to determine the mechanisms by which these poor metabolic outcomes arise, we investigated the impact of chronic phase shifts (CPS) on maternal and fetal hormonal, metabolic and circadian rhythms. We assessed weight gain and food consumption of dams exposed to either CPS or control lighting conditions throughout gestation. At day 20, dams were assessed for plasma hormone and metabolite concentrations and glucose and insulin tolerance. Additionally, the expression of a range of circadian and metabolic genes was assessed in maternal, placental and fetal tissue. Control and CPS dams consumed the same amount of food, yet CPS dams gained 70% less weight during the first week of gestation. At day 20, CPS dams had reduced retroperitoneal fat pad weight (-15%), and time-of-day dependent decreases in liver weight, whereas fetal and placental weight was not affected. Melatonin secretion was not altered, yet the timing of corticosterone, leptin, glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations were profoundly disrupted. The expression of gluconeogenic and circadian clock genes in maternal and fetal liver became either arrhythmic or were in antiphase to the controls. These results demonstrate that disruptions of the photoperiod can severely disrupt normal circadian profiles of plasma hormones and metabolites, as well as gene expression in maternal and fetal tissues. Disruptions in the timing of food consumption and the downstream metabolic processes required to utilise that food, may lead to reduced efficiency of growth such that maternal weight gain is reduced during early embryonic development. It is these perturbations that may contribute to the programming of poor metabolic homeostasis in the offspring.

Differential effects of exposure to maternal obesity or maternal weight loss during the periconceptional period in the sheep on insulin signalling molecules in skeletal muscle of the offspring at 4 months of age.

Exposure to maternal obesity before and/or throughout pregnancy may increase the risk of obesity and insulin resistance in the offspring in childhood and adult life, therefore, resulting in its transmission into subsequent generations. We have previously shown that exposure to maternal obesity around the time of conception alone resulted in increased adiposity in female lambs. Changes in the abundance of insulin signalling molecules in skeletal muscle and adipose tissue precede the development of insulin resistance and type 2 diabetes. It is not clear, however, whether exposure to maternal obesity results in insulin resistance in her offspring as a consequence of the impact of increased adiposity on skeletal muscle or as a consequence of the programming of specific changes in the abundance of insulin signalling molecules in this tissue. We have used an embryo transfer model in the sheep to investigate the effects of exposure to either maternal obesity or to weight loss in normal and obese mothers preceding and for one week after conception on the expression and abundance of insulin signalling molecules in muscle in the offspring. We found that exposure to maternal obesity resulted in lower muscle GLUT-4 and Ser 9 phospho-GSK3α and higher muscle GSK3α abundance in lambs when compared to lambs conceived in normally nourished ewes. Exposure to maternal weight loss in normal or obese mothers, however, resulted in lower muscle IRS1, PI3K, p110ß, aPKCζ, Thr 642 phospho-AS160 and GLUT-4 abundance in the offspring. In conclusion, maternal obesity or weight loss around conception have each programmed specific changes on subsets of molecules in the insulin signalling, glucose transport and glycogen synthesis pathways in offspring. There is a need for a stronger evidence base to ensure that weight loss regimes in obese women seeking to become pregnant minimize the metabolic costs for the next generation.

Maternal obesity and the early origins of childhood obesity: weighing up the benefits and costs of maternal weight loss in the periconceptional period for the offspring.

There is a need to understand the separate or interdependent contributions of maternal prepregnancy BMI, gestational weight gain, glycaemic control, and macronutrient intake on the metabolic outcomes for the offspring. Experimental studies highlight that there may be separate influences of maternal obesity during the periconceptional period and late gestation on the adiposity of the offspring. While a period of dietary restriction in obese mothers may ablate the programming of obesity, it is associated with an activation of the stress axis in the offspring. Thus, maternal obesity may result in epigenetic changes which predict the need for efficient fat storage in postnatal life, while maternal weight loss may lead to epigenetic changes which predict later adversity. Thus, development of dietary interventions for obese mothers during the periconceptional period requires a greater evidence base which allows the effective weighing up of the metabolic benefits and costs for the offspring.

Periconceptional undernutrition in normal and overweight ewes leads to increased adrenal growth and epigenetic changes in adrenal IGF2/H19 gene in offspring.

Adverse conditions in early life result in increased activation of the hypothalamo-pituitary-adrenal axis and in stress responsiveness in offspring. We have developed a model in which "donor" ewes are either normally nourished or overnourished prior to a period of dietary restriction, before transfer of the embryo at 6-7 d after conception to a ewe of normal weight and nutritional history. A moderate restriction of energy intake during the periconceptional period in both normal weight and overweight ewes resulted in increased adrenal mass in male and female lambs and an increased cortisol response to stress in female lambs. The increase in adrenal weight in lambs exposed to periconceptional undernutrition was associated with a decrease in the adrenal mRNA expression of IGF2 and decreased methylation in the proximal CTCF-binding site in the differentially methylated region of the IGF2/H19 gene. Thus, weight loss in both normal and overweight mothers during the periconceptional period results in epigenetic modification of IGF2 in the adrenal gland, adrenal overgrowth, and increased vulnerability to stress in offspring. Determining the appropriate approach to weight loss in the periconceptional period may therefore be important in overweight or obese women seeking to become pregnant.

The early origins of later obesity: pathways and mechanisms.

Excess bodyweight is the sixth most important risk factor contributing to the overall burden of disease worldwide. In excess of a billion adults and 10% of all children are now classified as overweight or obese. The main adverse consequences of obesity are the metabolic syndrome, cardiovascular disease and type 2 diabetes and a diminished average life expectancy. It has been argued that the complex pathological processes underlying obesity reflect environmental and genetic interactions, and individuals from disadvantaged communities seem to have greater risks than more affluent individuals partly because of fetal and postnatal programming interactions. Abundant evidence indicates that the obesity epidemic reflects progressive secular and age-related decreases in physical activity, together with passive over-consumption of energy dense foods despite neurobiological processes designed to regulate energy balance. The difficulty in treating obesity, however, highlights the deficits in our current understanding of the pathophysiology which underlies the initiation and chronic nature of this disorder. Large population based studies in Europe and North America in healthy women and in women with gestational diabetes have demonstrated that there are clear relationships between maternal and fetal nutrient supply, fetal growth patterns and the subsequent risk of obesity and glucose intolerance in childhood and adult life. In this review we discuss the impact of fetal nutrition on the biology of the developing adipocyte and brain and the growing evidence base supporting an intergenerational cycle of obesity.