Leptin-dependent differential remodeling of visceral and pericardial adipose tissue following chronic exercise and psychosocial stress.

Obesity is driven by an imbalance between caloric intake and energy expenditure, causing excessive storage of triglycerides in adipose tissue at different sites around the body. Increased visceral adipose tissue (VAT) is associated with diabetes, while pericardial adipose tissue (PAT) is associated with cardiac pathology. Adipose tissue can expand either through cellular hypertrophy or hyperplasia, with the former correlating with decreased metabolic health in obesity. The aim of this study was to determine how VAT and PAT remodel in response to obesity, stress, and exercise. Here we have used the male obese Zucker rats, which carries two recessive fa alleles that result in the development of hyperphagia with reduced energy expenditure, resulting in morbid obesity and leptin resistance. At 9 weeks of age, a group of lean (Fa/Fa or Fa/fa) Zucker rats (LZR) and obese (fa/fa) Zucker rats (OZR) were treated with unpredictable chronic mild stress or exercise for 8 weeks. To determine the phenotype for PAT and VAT, tissue cellularity and gene expression were analyzed. Finally, leptin signaling was investigated further using cultured 3T3-derived adipocytes. Tissue cellularity was determined following hematoxylin and eosin (H&E) staining, while qPCR was used to examine gene expression. PAT adipocytes were significantly smaller than those from VAT and had a more beige-like appearance in both LZR and OZR. In the OZR group, VAT adipocyte cell size increased significantly compared with LZR, while PAT showed no difference. Exercise and stress resulted in a significant reduction in VAT cellularity in OZR, while PAT showed no change. This suggests that PAT cellularity does not remodel significantly compared with VAT. These data indicate that the extracellular matrix of PAT is able to remodel more readily than in VAT. In the LZR group, exercise increased insulin receptor substrate 1 (IRS1) in PAT but was decreased in the OZR group. In VAT, exercise decreased IRS1 in LZR, while increasing it in OZR. This suggests that in obesity, VAT is more responsive to exercise and subsequently becomes less insulin resistant compared with PAT. Stress increased PPAR-γ expression in the VAT but decreased it in the PAT in the OZR group. This suggests that in obesity, stress increases adipogenesis more significantly in the VAT compared with PAT. To understand the role of leptin signaling in adipose tissue remodeling mechanistically, JAK2 autophosphorylation was inhibited using 5 µM 1,2,3,4,5,6-hexabromocyclohexane (Hex) in cultured 3T3-derived adipocytes. Palmitate treatment was used to induce cellular hypertrophy. Hex blocked adipocyte hypertrophy in response to palmitate treatment but not the increase in lipid droplet size. These data suggest that leptin signaling is necessary for adipocyte cell remodeling, and its absence induces whitening. Taken together, our data suggest that leptin signaling is necessary for adipocyte remodeling in response to obesity, exercise, and psychosocial stress.

Intracellular and Tissue Levels of Vitamin B12 in Hepatocytes Are Modulated by CD320 Receptor and TCN2 Transporter.

The liver mass constitutes hepatocytes expressing receptors for vitamin B12 (B12)-bound transporters in circulation. However, intrahepatic and circulating B12 interrelationship levels remain unclear. We assessed the intracellular B12 levels at various circulating B12 concentrations in human HepG2 cell-line and liver tissue levels of B12 in the C57BL/6 mouse model. In HepG2 cells treated with a range of B12 concentrations, the intracellular and circulatory B12 levels, transcript and protein levels of B12 receptor (CD320) and transporter (TCN2) were determined using immunoassays, qRT-PCR and Western blot, respectively. Similar assessments were done in plasma and liver tissue of C57BL/6 mice, previously fed a diet of either a high or low B12 (30.82 µg B12/kg and 7.49 µg B12/kg, respectively) for 8-10 weeks. The physiological B12 status (0.15-1 nM) resulted in increased levels of intracellular B12 in HepG2 cells compared to supraphysiological levels of B12 (>1 nM). Gene and protein expression of CD320 and TCN2 were also higher at physiological levels of B12. Progressively increasing extracellular B12 to supraphysiological levels led to relative decreased levels of intracellular B12, lower expression of gene and protein levels of CD320 and TCN2. Similar results were observed in liver tissue from mice fed on a low B12 diet verses high B12 diet. These findings suggest that unlike supraphysiological B12, physiological levels of B12 in the extracellular media or circulation accelerates active transport of B12, and expression of CD320 and TCN2, resulting in higher relative uptake of B12 in hepatocytes.

Maternal Obesity during Pregnancy Alters Daily Activity and Feeding Cycles, and Hypothalamic Clock Gene Expression in Adult Male Mouse Offspring.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

Altered cellular redox status, sirtuin abundance and clock gene expression in a mouse model of developmentally primed NASH.

BACKGROUND: We have previously shown that high fat (HF) feeding during pregnancy primes the development of non-alcoholic steatohepatits (NASH) in the adult offspring. However, the underlying mechanisms are unclear. AIMS: Since the endogenous molecular clock can regulate hepatic lipid metabolism, we investigated whether exposure to a HF diet during development could alter hepatic clock gene expression and contribute to NASH onset in later life. METHODS: Female mice were fed either a control (C, 7%kcal fat) or HF (45%kcal fat) diet. Offspring were fed either a C or HF diet resulting in four offspring groups: C/C, C/HF, HF/C and HF/HF. NAFLD progression, cellular redox status, sirtuin expression (Sirt1, Sirt3), and the expression of core clock genes (Clock, Bmal1, Per2, Cry2) and clock-controlled genes involved in lipid metabolism (Rev-Erbα, Rev-Erbß, RORα, and Srebp1c) were measured in offspring livers. RESULTS: Offspring fed a HF diet developed NAFLD. However HF fed offspring of mothers fed a HF diet developed NASH, coupled with significantly reduced NAD(+)/NADH (p<0.05, HF/HF vs C/C), Sirt1 (p<0.001, HF/HF vs C/C), Sirt3 (p<0.01, HF/HF vs C/C), perturbed clock gene expression, and elevated expression of genes involved lipid metabolism, such as Srebp1c (p<0.05, C/HF and HF/HF vs C/C). CONCLUSION: Our results suggest that exposure to excess dietary fat during early and post-natal life increases the susceptibility to develop NASH in adulthood, involving altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression.

Fetal programming of adipose tissue function: an evolutionary perspective.

Obesity is an escalating threat of pandemic proportions and has risen to such unrivaled prominence in such a short period of time that it has come to define a whole generation in many countries around the globe. The burden of obesity, however, is not equally shared among the population, with certain ethnicities being more prone to obesity than others, while some appear to be resistant to obesity altogether. The reasons behind this ethnic basis for obesity resistance and susceptibility, however, have remained largely elusive. In recent years, much evidence has shown that the level of brown adipose tissue thermogenesis, which augments energy expenditure and is negatively associated with obesity in both rodents and humans, varies greatly between ethnicities. Interestingly, the incidence of low birth weight, which is associated with an increased propensity for obesity and cardiovascular disease in later life, has also been shown to vary by ethnic background. This review serves to reconcile ethnic variations in BAT development and function with ethnic differences in birth weight outcomes to argue that the variation in obesity susceptibility between ethnic groups may have its origins in the in utero programming of BAT development and function as a result of evolutionary adaptation to cold environments.

Effect of maternal protein restriction during pregnancy and postweaning high-fat feeding on diet-induced thermogenesis in adult mouse offspring.

PURPOSE: Prenatal undernutrition followed by postweaning feeding of a high-fat diet results in obesity in the adult offspring. In this study, we investigated whether diet-induced thermogenesis is altered as a result of such nutritional mismatch. METHODS: Female MF-1 mice were fed a normal protein (NP, 18% casein) or a protein-restricted (PR, 9% casein) diet throughout pregnancy and lactation. After weaning, male offspring of both groups were fed either a high-fat diet (HF; 45% kcal fat) or standard chow (C, 7% kcal fat) to generate the NP/C, NP/HF, PR/C and PR/HF adult offspring groups (n = 7-11 per group). RESULTS: PR/C and NP/C offspring have similar body weights at 30 weeks of age. Postweaning HF feeding resulted in significantly heavier NP/HF offspring (P < 0.01), but not in PR/HF offspring, compared with their chow-fed counterparts. However, the PR/HF offspring exhibited greater adiposity (P < 0.01) v the NP/HF group. The NP/HF offspring had increased energy expenditure and increased mRNA expression of uncoupling protein-1 and ß-3 adrenergic receptor in the interscapular brown adipose tissue (iBAT) compared with the NP/C mice (both at P < 0.01). No such differences in energy expenditure and iBAT gene expression were observed between the PR/HF and PR/C offspring. CONCLUSIONS: These data suggest that a mismatch between maternal diet during pregnancy and lactation, and the postweaning diet of the offspring, can attenuate diet-induced thermogenesis in the iBAT, resulting in the development of obesity in adulthood.

The housekeeping gene YWHAZ remains stable in a model of developmentally primed non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in Western society. Comparative gene expression studies are beginning to elucidate the molecular mechanisms underlying NAFLD progression. We have previously shown that high fat diets during early life can prime non-alcoholic steatohepatitis (NASH) in adulthood, through lipogenesis gene elevation. To generate accurate results in such studies, appropriate housekeeping genes (HKG) which are unaffected by disease processes, are used for data normalisation. However, there is little existing data to show the effects of NAFLD on HKG expression. AIMS: To identify the HKG in a mouse model of developmentally primed NAFLD and NASH, which maintains expression stability. METHODS: We determined the expression stability of six candidates HKG (GAPDH, YWHAZ, B2M, EIF4A2, ACTB and CYC1) in a mouse model of developmentally primed NAFLD in both the day and night, using geNORM qBasePlus software. RESULTS: HKG expression differed across dietary groups and time of day. In the majority of treatment groups and time points the most stable gene was YWHAZ. Following high fat diet interventions CYC1 became notably unstable. Overall the effect of NAFLD and NASH on HKG expression was to maintain stability of YWHAZ, but destabilise CYC1 and EIF4A2. CONCLUSIONS: Our data clearly shows that HKG expression is affected by NAFLD severity and time of day sampling, highlighting the importance of suitable HKG gene selection. For comparative gene expression studies investigating NAFLD we would recommend use of YWHAZ as a robust, stably expressed HKG.

The role of the circadian clock system in nutrition and metabolism.

Mammals have an endogenous timing system in the suprachiasmatic nuclei (SCN) of the hypothalamic region of the brain. This internal clock system is composed of an intracellular feedback loop that drives the expression of molecular components and their constitutive protein products to oscillate over a period of about 24 h (hence the term 'circadian'). These circadian oscillations bring about rhythmic changes in downstream molecular pathways and physiological processes such as those involved in nutrition and metabolism. It is now emerging that the molecular components of the clock system are also found within the cells of peripheral tissues, including the gastrointestinal tract, liver and pancreas. The present review examines their role in regulating nutritional and metabolic processes. In turn, metabolic status and feeding cycles are able to feed back onto the circadian clock in the SCN and in peripheral tissues. This feedback mechanism maintains the integrity and temporal coordination between various components of the circadian clock system. Thus, alterations in environmental cues could disrupt normal clock function, which may have profound effects on the health and well-being of an individual.

A High Fat "Western-style" Diet Induces AMD-Like Features in Wildtype Mice.

SCOPE: The intake of a "Western-style" diet rich in fats is linked with developing retinopathies including age-related macular degeneration (AMD). Wildtype mice are given a high fat diet (HFD) to determine how unhealthy foods can bring about retinal degeneration. METHODS AND RESULTS: Following weaning, female C57BL/6 mice are maintained on standard chow (7% kcal fat, n = 29) or a HFD (45% kcal fat, n = 27) for 12 months. Animals were sacrificed following electroretinography (ERG) and their eyes analyzed by histology, confocal immunofluorescence, and transmission electron microscopy. HFD mice become obese, but showed normal retinal function compared to chow-fed controls. However, diminished ß3tubulin labeling of retinal cross-sections indicated fewer/damaged neuronal processes in the inner plexiform layer. AMD-linked proteins clusterin and TIMP3 accumulated in the retinal pigment epithelium (RPE) and Bruch's membrane (BrM). Neutral lipids also deposited in the outer retinae of HFD mice. Ultrastructural analysis revealed disorganized photoreceptor outer segments, collapsed/misaligned RPE microvilli, vacuoles, convoluted basolateral RPE infolds and BrM changes. Basal laminar-like deposits were also present alongside abnormal choroidal endothelial cells. CONCLUSIONS: We show that prolonged exposure to an unhealthy "Western-style" diet alone can recapitulate early-intermediate AMD-like features in wildtype mice, highlighting the importance of diet and nutrition in the etiology of sight-loss.

Developmental origins of the metabolic syndrome: body clocks and stress responses.

The prevalence of the metabolic syndrome, which represents a spectrum of metabolic and cardiovascular disorders, continues to increase at an alarming rate in contemporary society. Inadequate responses of an individual to environmental challenges such as unbalanced diet or lack of physical exercise during their life course has been recognised to increase risk of this pathological condition. Recent evidence suggests that this may involve alterations in the settings of the circadian clock system, which consists of oscillating molecular pacemakers found not only in the hypothalamic region of the brain but also in most peripheral tissues, and of the hypothalamic-pituitary-adrenal (HPA) axis which regulates stress responses. These two systems are now known to interact to produce an integrated response to environmental challenges. In this review, we highlight the importance of environmental cues during early development in establishing the homeostatic set-points of the circadian clock and HPA stress systems. These effects can operate within the normal range and are not in themselves pathological, but can nevertheless affect an individual's response to environmental challenges in adult life and thus their risk of the metabolic syndrome.

Epigenetic priming of the metabolic syndrome.

The metabolic syndrome (MetS) represents a cluster of cardiometabolic risk factors, including central obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, hyperinsulinemia and microalbuminuria, and more recently, nonalcoholic fatty liver disease (NAFLD), polycystic ovarian syndrome (PCOS) and atherosclerosis. Although the concept of the MetS is subject to debate due to lack of a unifying underlying mechanism, the prevalence of a metabolic syndrome phenotype is rapidly increasing worldwide. Moreover, it is increasingly prevalent in children and adolescents of obese mothers. Evidence from both epidemiological and experimental animal studies now demonstrates that MetS onset is increasingly likely following exposure to suboptimal nutrition during critical periods of development, as observed in maternal obesity. Thus, the developmental priming of the MetS provides a common origin for this multifactorial disorder. Consequently, the mechanisms leading to this developmental priming have recently been the subject of intensive investigation. This review discusses recent data regarding the epigenetic modifications resulting from nutrition during early development that mediate persistent changes in the expression of key metabolic genes and contribute toward an adult metabolic syndrome phenotype. In addition, this review considers the role of the endogenous molecular circadian clock system, which has the potential to act at the interface between nutrient sensing and epigenetic processing. A continued and greater understanding of these mechanisms will eventually aid in the identification of individuals at high risk of cardiovascular disease (CVD) and type 2 diabetes, and help develop therapeutic interventions, in accordance with current global government strategy.

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

UNLABELLED: 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.

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

BACKGROUND: miRNAs 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. RESULTS: We 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. CONCLUSION: Maternal 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.

Metabolic disturbances in non-alcoholic fatty liver disease.

NAFLD (non-alcoholic fatty liver disease) refers to a wide spectrum of liver damage, ranging from simple steatosis to NASH (non-alcoholic steatohepatitis), advanced fibrosis and cirrhosis. NAFLD is strongly associated with insulin resistance and is defined by accumulation of liver fat >5% per liver weight in the presence of <10 g of daily alcohol consumption. The exact prevalence of NAFLD is uncertain because of the absence of simple non-invasive diagnostic tests to facilitate an estimate of prevalence. In certain subgroups of patients, such as those with Type 2 diabetes, the prevalence of NAFLD, defined by ultrasound, may be as high as 70%. NASH is an important subgroup within the spectrum of NAFLD that progresses over time with worsening fibrosis and cirrhosis, and is associated with increased risk for cardiovascular disease. It is, therefore, important to understand the pathogenesis of NASH and, in particular, to develop strategies for interventions to treat this condition. Currently, the 'gold standard' for the diagnosis of NASH is liver biopsy, and the need to undertake a biopsy has impeded research in subjects in this field. Limited results suggest that the prevalence of NASH could be as high as 11% in the general population, suggesting there is a worsening future public health problem in this field of medicine. With a burgeoning epidemic of diabetes in an aging population, it is likely that the prevalence of NASH will continue to increase over time as both factors are important risk factors for liver fibrosis. The purpose of this review is to: (i) briefly discuss the epidemiology of NAFLD to describe the magnitude of the future potential public health problem; and (ii) to discuss extra- and intra-hepatic mechanisms contributing to the pathogenesis of NAFLD, a better understanding of which may help in the development of novel treatments for this condition.

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 Obesity During Pregnancy and Lactation Influences Offspring Obesogenic Adipogenesis but Not Developmental Adipogenesis in Mice.

Obesity is an escalating health crisis of pandemic proportions and by all accounts it has yet to reach its peak. Growing evidence suggests that obesity may have its origins in utero. Recent studies have shown that maternal obesity during pregnancy may promote adipogenesis in offspring. However, these studies were largely based on cell culture models. Whether or not maternal obesity impacts on offspring adipogenesis in vivo remains to be fully established. Furthermore, in vivo adipogenic differentiation has been shown to happen at distinct time periods, one during development (developmental adipogenesis-which is complete by 4 weeks of age in mice) and another in adulthood in response to feeding a high-fat (HF) diet (obesogenic adipogenesis). We therefore set out to determine whether maternal obesity impacted on offspring adipocyte hyperplasia in vivo and whether maternal obesity impacted on developmental or obesogenic adipogenesis, or both. Our findings reveal that maternal obesity is associated with enhanced obesogenic adipogenesis in HF-fed offspring. Interestingly, in newly weaned (4-week-old) offspring, maternal obesity is associated with adipocyte hypertrophy, but there were no changes in adipocyte number. Our results suggest that maternal obesity impacts on offspring obesogenic adipogenesis but does not affect developmental adipogenesis.

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.

Appetite regulatory mechanisms and food intake in mice are sensitive to mismatch in diets between pregnancy and postnatal periods.

Human and animal studies suggest that obesity in adulthood may have its origins partly during prenatal development. One of the underlying causes of obesity is the perturbation of hypothalamic mechanisms controlling appetite. We determined mRNA levels of genes that regulate appetite, namely neuropeptide Y (NPY), pro-opiomelanocortin (POMC) and the leptin receptor isoform Ob-Rb, in the hypothalamus of adult mouse offspring from pregnant dams fed a protein-restricted diet, and examined whether mismatched post-weaning high-fat diet altered further expression of these gene transcripts. Pregnant MF1 mice were fed either normal protein (C, 18% casein) or protein-restricted (PR, 9% casein) diet throughout pregnancy. Weaned offspring were fed to adulthood a high-fat (HF; 45% kcal fat) or standard chow (21% kcal fat) diet to generate the C/HF, C/C, PR/HF and PR/C groups. Food intake and body weight were monitored during this period. Hypothalamic tissues were collected at 16 weeks of age for analysis of gene expression by real time RT-PCR. All HF-fed offspring were observed to be heavier vs. C groups regardless of the maternal diet during pregnancy. In the PR/HF males, but not in females, daily energy intake was reduced by 20% vs. the PR/C group (p<0.001). In PR/HF males, hypothalamic mRNA levels were lower vs. the PR/C group for NPY (p<0.001) and Ob-Rb (p<0.05). POMC levels were similar in all groups. In females, mRNA levels for these transcripts were similar in all groups. Our results suggest that adaptive changes during prenatal development in response to maternal dietary manipulation may have long-term sex-specific consequences on the regulation of appetite and metabolism following post-weaning exposure to an energy-rich nutritional environment.

Sensitivity of housekeeping genes in the hypothalamus to mismatch in diets between pre- and postnatal periods in mice.

Housekeeping genes are used as internal controls in gene expression studies, but their expression levels vary according to tissue types and experimental treatments. A nutritional mismatch between pre- and postnatal periods, wherein the in utero nutritional environment is suboptimal and post-weaning diet is rich in fat, results in altered hypothalamic expression levels of genes that regulate the offspring's physiology, metabolism and behavior. The present study investigated hypothalamic expression of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin and 18s ribosomal RNA (18s rRNA) in offspring subjected to this pre- and postnatal dietary mismatch. Pregnant MF1 mice were fed standard chow (C, 18% casein) or protein restricted (PR, 9% casein) diet throughout pregnancy. Weaned offspring were fed to adulthood a high fat (HF, 45% kcal fat) or chow (21% kcal fat) diet to generate the C/HF, C/C, PR/HF and PR/C groups. Hypothalamic and cerebral cortex tissues were collected from these offspring at 16 weeks of age and analyzed for gene transcript levels by quantitative real time PCR. Hypothalamic GAPDH mRNA levels were higher in PR/HF male and female offspring vs. all other groups (p<0.001 in males). Conversely, hypothalamic beta-actin and 18s rRNA levels were similar in all treatment groups and sex. In the cerebral cortex, GAPDH and beta-actin levels were similar in all groups and sex. The result suggests that beta-actin and 18s rRNA are suitable internal controls for gene expression studies in the hypothalamus, while the stability of GAPDH is compromised, under the condition of a nutritional mismatch between pre- and postnatal periods.

The Divergent Effect of Maternal Protein Restriction during Pregnancy and Postweaning High-Fat Diet Feeding on Blood Pressure and Adiposity in Adult Mouse Offspring.

Obesity is a growing health crisis of pandemic proportions. Numerous animal and human studies have confirmed that obesity and related metabolic abnormalities, such as insulin resistance and cardiovascular disease, may be programmed during development by adverse maternal nutrition. We previously documented that offspring of female mice who were protein-restricted during pregnancy alone had no alterations to their body weights, but did display a considerable reduction in food intake, a finding which was linked to reduced expression levels of appetite regulatory genes in the hypothalamus. Whether such observations were accompanied by changes in metabolic and phenotypic parameters remained to be determined. Female pregnant MF-1 mice were fed, exclusively during the pregnancy period, a normal protein diet containing 18% casein (C) or an isocaloric protein-restricted diet containing 9% casein (PR). From birth, the lactating dams were fed a normal protein diet. At weaning, offspring were fed either the standard chow which contain 7% kcal fat (C) or high-fat diet (HF, 45% kcal fat). This yielded 4 experimental groups denoted by maternal diet/offspring diet: C/C, C/HF, PR/C, PR/HF. Our results showed that offspring adiposity was significantly increased in HF-fed offspring, and was not affected by the 50% reduction in protein content of the maternal diet fed during pregnancy. Similarly, blood glucose levels were higher in HF-fed offspring, regardless of protein content of the maternal diet. Systolic blood pressure, on the other hand, was significantly increased in both male and female offspring of dams fed the PR diet, and this was exacerbated by a postweaning HF diet. Our results show that maternal protein restriction leads to elevations in systolic blood pressure, which is exacerbated by a postweaning HF-diet. Our present findings suggest that, while changes in offspring adiposity brought about by exposure to maternal protein restriction during pregnancy may be restored by adequate maternal protein content during lactation, the same may not be true for systolic blood pressure, which was similarly impaired, regardless of the timing of maternal low-protein exposure.