Roselle L. Cripps

Maternal diet influences DNA damage, aortic telomere length, oxidative stress, and antioxidant defense capacity in rats

Low birth weight is associated with in creased cardiovascular disease (CVD) in humans. Detri mental effects of low birth weight are amplified by rapid catch–up growth. Conversely, slow growth during lactation reduces CVD risk. Gestational protein restriction causes low birth weight, vascular dysfunction, and accelerated aging in rats. Atherosclerotic aortic tissue has shortened telomeres, and oxidative stress accelerates telomere short ening through generation of DNA single–strand breaks (ssbs). This study tested the hypothesis that maternal diet influences aortic telomere length through changes in DNA ssbs, antioxidant capacity, and oxidative stress. We used our models of gestational protein restriction followed by rapid catch–up growth (the recuperated group) and pro tein restriction during lactation (the postnatal low–protein [PLP] group). Southern blotting revealed fewer aortic DNA ssbs and subsequently fewer short telomeres (P<0.05) in the PLP group. This result was associated with reduced (P<0.01) 8–hydroxy–2–deoxyguanosine, a marker of oxidative stress. PLP animals expressed in creased (P<0.01) manganese superoxide–dismutase, cop per–zinc superoxide–dismutase, catalase, and glutathione– reductase. Age–dependent changes in antioxidant defense enzymes indicated more protection to oxidative stress in the PLP animals;conversely, recuperated animals demon strated age–associated impairment of antioxidant de fenses. We conclude that maternal diet has a major influence on aortic telomere length. This finding may provide a mechanistic link between early growth patterns and CVD.— Tarry‐Adkins, J. L., Martin‐Gronert, M. S., Chen, J.‐H., Cripps, R. L., Ozanne, S. E. Maternal diet influences DNA damage, aortic telomere length, oxidative stress, and antioxidant defense capacity in rats. FASEB J. 22, 2037–2044 (2008)

Fetal and perinatal programming of appetite

There is increasing concern about the rapidly rising incidence of obesity worldwide and its impact both on mortality, morbidity and the cost of healthcare. In the last 15 years, a large volume of research has linked low birth weight to many adult diseases in humans, such as Type II diabetes, cardiovascular disease, hypertension and the metabolic syndrome. Obesity is a causal factor in all these conditions. There are epidemiological studies linking low birth weight to increased adiposity, but the timing of the insult during gestation seems crucial, as reducing maternal nutrition in late gestation and during lactation causes a reduction in later obesity. Recent studies in animal models have provided clues towards mechanisms of altered appetite regulation following alterations in fetal and neonatal growth. The outcome of these and future studies could prove clinically crucial, particularly in the debate over the benefits of breast feeding, which provides a lower plane of nutrition compared with formula feeding.

Developmental changes in hypothalamic leptin receptor: relationship with the postnatal leptin surge and energy balance neuropeptides in the postnatal rat

In the adult brain, leptin regulates energy homeostasis primarily via hypothalamic circuitry that affects food intake and energy expenditure. Evidence from rodent models has demonstrated that during early postnatal life, leptin is relatively ineffective in modulating these pathways, despite the high circulating levels and the presence of leptin receptors within the central nervous system. Furthermore, in recent years, a neurotrophic role for leptin in the establishment of energy balance circuits has emerged. The precise way in which leptin exerts these effects, and the site of leptin action, is unclear. To provide a detailed description of the development of energy balance systems in the postnatal rat in relation to leptin concentrations during this time, endogenous leptin levels were measured, along with gene expression of leptin receptors and energy balance neuropeptides in the medial basal hypothalamus, using in situ hybridization. Expression of leptin receptors and both orexigenic and anorexigenic neuropeptides increased in the arcuate nucleus during the early postnatal period. At postnatal day 4 (P4), we detected dense leptin receptor expression in ependymal cells of the third ventricle (3V), which showed a dramatic reduction over the first postnatal weeks, coinciding with marked morphological changes in this region. An acute leptin challenge robustly induced suppressor of cytokine signaling-3 expression in the 3V of P4 but not P14 animals, revealing a clear change in the location of leptin action over this period. These findings suggest that the neurotrophic actions of leptin may involve signaling at the 3V during a restricted period of postnatal development.

Catch-up growth following intra-uterine growth-restriction programmes an insulin-resistant phenotype in adipose tissue

Background:It is now widely accepted that the early-life nutritional environment is important in determining susceptibility to metabolic diseases. In particular, intra-uterine growth restriction followed by accelerated postnatal growth is associated with an increased risk of obesity, type-2 diabetes and other features of the metabolic syndrome. The mechanisms underlying these observations are not fully understood.Aim:Using a well-established maternal protein-restriction rodent model, our aim was to determine if exposure to mismatched nutrition in early-life programmes adipose tissue structure and function, and expression of key components of the insulin-signalling pathway.Methods:Offspring of dams fed a low-protein (8%) diet during pregnancy were suckled by control (20%)-fed dams to drive catch-up growth. This ‘recuperated’ group was compared with offspring of dams fed a 20% protein diet during pregnancy and lactation (control group). Epididymal adipose tissue from 22-day and 3-month-old control and recuperated male rats was studied using histological analysis. Expression and phosphorylation of insulin-signalling proteins and gene expression were assessed by western blotting and reverse-transcriptase PCR, respectively.Results:Recuperated offspring at both ages had larger adipocytes (P<0.001). Fasting serum glucose, insulin and leptin levels were comparable between groups but increased with age. Recuperated offspring had reduced expression of IRS-1 (P<0.01) and PI3K p110β (P<0.001) in adipose tissue. In adult recuperated rats, Akt phosphorylation (P<0.01) and protein levels of Akt-2 (P<0.01) were also reduced. Messenger RNA expression levels of these proteins were not different, indicating a post-transcriptional effect.Conclusion:Early-life nutrition programmes alterations in adipocyte cell size and impairs the protein expression of several insulin-signalling proteins through post-transcriptional mechanisms. These indices may represent early markers of insulin resistance and metabolic disease risk.

Programming of hypothalamic neuropeptide gene expression in rats by maternal dietary protein content during pregnancy and lactation.

Epidemiological studies show a link between low birthweight and increased obesity. In contrast, slow growth during the lactation period reduces obesity risk. The present study investigates the potential underlying mechanisms of these observations. Rats were established as follows: (i) control animals [offspring of control dams fed a 20% (w/v) protein diet], (ii) recuperated animals [offspring of dams fed an isocaloric low-protein (8%, w/v) diet during pregnancy and nursed by control dams], and (iii) postnatal low protein animals (offspring of control dams nursed by low-protein-fed dams). Serum and brains were collected from fed and fasted animals at weaning. Expression of hypothalamic energy balance genes was assessed using in situ hybridization. Recuperated pups were smaller at birth, but caught up with controls by day 21 and gained more weight than controls between weaning and 12 weeks of age (P<0.05). At 21 days, they were hypoleptinaemic compared with controls in the fed state, with generally comparable hypothalamic gene expression. Postnatal low protein offspring had significantly lower body weights than controls at weaning and 12 weeks of age (P<0.001). At 21 days, they were hypoglycaemic, hypoinsulinaemic and hypoleptinaemic. Leptin receptor gene expression in the arcuate nucleus was increased in postnatal low protein animals compared with controls. Consistent with hypoleptinaemia, hypothalamic gene expression for the orexigenic neuropeptides NPY (neuropeptide Y) and AgRP (Agouti-related peptide) was increased, and that for the anorexigenic neuropeptides POMC (pro-opiomelanocortin) and CART (cocaine- and amphetamine-regulated transcript) was decreased. These results suggest that the early nutritional environment can affect the development of energy balance circuits and consequently obesity risk.

Role of leptin in the regulation of growth and carbohydrate metabolism in the ovine fetus during late gestation

Leptin is an important regulator of appetite and energy expenditure in adulthood, although its role as a nutritional signal in the control of growth and metabolism before birth is poorly understood. This study investigated the effects of leptin on growth, carbohydrate metabolism and insulin signalling in fetal sheep. Crown–rump length‐measuring devices and vascular catheters were implanted in 12 sheep fetuses at 105–110 days of gestation (term 145 ± 2 days). The fetuses were infused i.v. either with saline (0.9% NaCl; n= 6) or recombinant ovine leptin (0.5–1.0 mg kg−1 day−1; n= 6) for 5 days from 125 to 130 days when they were humanely killed and tissues collected. Leptin receptor mRNA and protein were expressed in fetal liver, skeletal muscle and perirenal adipose tissue. Throughout infusion, plasma leptin in the leptin‐infused fetuses was 3‐ to 5‐fold higher than in the saline‐infused fetuses, although plasma concentrations of insulin, glucose, lactate, cortisol, catecholamines and thyroid hormones did not differ between the groups. Leptin infusion did not affect linear skeletal growth or body, placental and organ weights in utero. Hepatic glycogen content and activities of the gluconeogenic enzymes glucose‐6‐phosphatase and phosphoenolpyruvate carboxykinase in the leptin‐infused fetuses were lower than in the saline‐infused fetuses by 44, 48 and 36%, respectively; however, there were no differences in hepatic glycogen synthase activity or insulin signalling protein levels. Therefore, before birth, leptin may inhibit endogenous glucose production by the fetal liver when adipose energy stores and transplacental nutrient delivery are sufficient for the metabolic needs of the fetus. These actions of leptin in utero may contribute to the development of neonatal hypoglycaemia in macrosomic babies of diabetic mothers.

Effects of Cortisol and Dexamethasone on Insulin Signalling Pathways in Skeletal Muscle of the Ovine Fetus during Late Gestation

Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.

Leanness in postnatally nutritionally programmed rats is associated with increased sensitivity to leptin and a melanocortin receptor agonist and decreased sensitivity to neuropeptide Y

Background:Pups of normally nourished dams that are cross-fostered after birth to dams fed a low-protein (8% by weight) diet (postnatal low protein (PLP)) grow slower during the suckling period and remain small and lean throughout adulthood. At weaning, they have increased expression in the arcuate nucleus (ARC) of the hypothalamus of the orexigenic neuropeptide Y (NPY) and decreased expression of pro-opiomelanocortin, the precursor of anorexigenic melanocortins.Objectives And Methods:We investigated, using third ventricle administration, whether 3-month-old male PLP rats display altered sensitivity to leptin with respect to food intake, NPY and the melanocortin 3/4-receptor agonist MTII, and using in situ hybridization or laser capture microdissection of the ARC followed by RT-PCR, whether the differences observed were associated with changes in the hypothalamic expression of NPY or the leptin receptor, NPY receptors and melanocortin receptors.Results:PLP rats were smaller and had reduced percentage body fat content and plasma leptin concentration compared with control rats. Leptin (5 μg) reduced food intake over 0–48 h more in PLP than control rats (P<0.05). Submaximal doses of NPY increased the food intake less in PLP rats than in controls, whereas submaximal doses of MTII reduced the food intake more in PLP rats. Maximal responses did not differ between PLP and control rats. Leptin and melanocortin-3 receptor (MC3R) expression were increased in both ARC and ventromedial hypothalamic nuclei in PLP animals compared with the controls. MC4R, NPY Y1R, Y5R and NPY expression were unchanged.Conclusion:Postnatal undernourishment results in food intake in adult rats being more sensitive to reduction by leptin and melanocortins, and less sensitive to stimulation by NPY. We propose that this contributes to increased leptin sensitivity and resistance to obesity. Increased expression of ObRb and MC3R may partly explain these findings but other downstream mechanisms must also be involved.

The Effect of Maternal Body Condition Score Before and During Pregnancy on the Glucose Tolerance of Adult Sheep Offspring

This study investigates the effects of diet-induced changes in maternal body condition on glucose tolerance in sheep. Welsh Mountain ewes were established, by dietary manipulation, at a body condition score of 2 (lower body condition [LBCS], n = 17) or >3 (higher body condition [HBCS], n = 19) prior to and during pregnancy. Birth weight and postnatal growth were similar in LBCS and HBCS offspring. In young adulthood, LBCS offspring had increased fasting glucose levels (3.8 ± 0.07 vs 3.6 ± 0.05 mM, P < .05), poorer glucose tolerance (2274 ± 22.6 vs 2161 ± 33 min/mM, P < .01), and reduced insulin secretion (0.58 ± 0.05 vs 0.71 ± 0.07 nM/min, P = .07). Increased fasting glycemia, mild glucose intolerance, and impaired initial insulin secretory response, as observed in LBCS offspring, are indictors of increased diabetes risk in humans. These findings suggest that altered maternal body composition and an imbalance between the fetal and postnatal environment influence offspring glucose tolerance.

Lower maternal body condition during pregnancy affects skeletal muscle structure and glut-4 protein levels but not glucose tolerance in mature adult sheep.

Suboptimal maternal nutrition and body composition are implicated in metabolic disease risk in adult offspring. We hypothesized that modest disruption of glucose homeostasis previously observed in young adult sheep offspring from ewes of a lower body condition score (BCS) would deteriorate with age, due to changes in skeletal muscle structure and insulin signaling mechanisms. Ewes were fed to achieve a lower (LBCS, n = 10) or higher (HBCS, n = 14) BCS before and during pregnancy. Baseline plasma glucose, glucose tolerance and basal glucose uptake into isolated muscle strips were similar in male offspring at 210 ± 4 weeks. Vastus total myofiber density (HBCS, 343 ± 15; LBCS, 294 ± 14 fibers/mm2, P < .05) and fast myofiber density (HBCS, 226 ± 10; LBCS 194 ± 10 fibers/mm2, P < .05), capillary to myofiber ratio (HBCS, 1.5 ± 0.1; LBCS 1.2 ± 0.1 capillary:myofiber, P < .05) were lower in LBCS offspring. Vastus protein levels of Akt1 were lower (83% ± 7% of HBCS, P < .05), and total glucose transporter 4 was increased (157% ± 6% of HBCS, P < .001) in LBCS offspring, Despite the reduction in total myofiber density in LBCS offspring, glucose tolerance was normal in mature adult life. However, such adaptations may lead to complications in metabolic control in an overabundant postnatal nutrient environment.