Markers of prenatal metabolic plasticity and assessment of their reversibility by postnatal interventions

Project Information

METABOLIC PLASTICITY

Grant agreement ID: 253131

Closed project

Start date 1 July 2010

End date 30 June 2013

Funded under

• Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Total cost € 238 375,30

EU contribution € 238 375,30

Coordinated by

IDRYMA IATROVIOLOGIKON EREUNON AKADEMIAS ATHINON
Greece

Final Report Summary - METABOLIC PLASTICITY (Markers of prenatal metabolic plasticity and assessment of their reversibility by postnatal interventions)

MARIE CURIE ACTIONS - International Outgoing Fellowships (IOF)
FP7-PEOPLE-2009-IOF “Metabolic Plasticity” GA 253131
Title: Markers of prenatal metabolic plasticity and assessment of their reversibility by postnatal interventions.
Contact information: Aristides Lytras, MD, Ph.D. (Marie Curie International Outgoing Fellow)
[e-mail]; [e-mail]; [e-mail]

Introduction: Inactivity and suboptimal nutrition have resulted in a growing population of individuals with adiposity, obesity and compromised whole-body metabolism, including insulin resistance. An increasing percentage of this pool of individuals develops overt type 2 diabetes (T2D), leading to vast personal and social burdens. A key emerging concept is that risk for diabetes may begin early in life, during both intrauterine and early postnatal development. In this context, metabolic stress during these critical developmental periods has a significant impact on the long-term organization of metabolic adaptive responses. Indeed, multiple human epidemiological studies have demonstrated that exposure to either overnutrition, undernutrition or other stressors during prenatal life appears to “program” susceptibility to T2D, through as yet unidentified mechanisms. It is thus critical to identify individuals at highest risk for developing diabetes in order to develop improved early intervention strategies for targeted prevention.
The challenge: Given the importance of understanding the pathophysiology of early diabetes risk, multiple in utero models have been developed, including low birth weight (LBW) due to maternal undernutrition (UN), maternal overnutrition, and isolated maternal insulin resistance (in utero exposure to maternal insulin resistance, without hyperglycemia or obesity; IUIR), among others. These constitute “metabolic” rather than “genetic” models of insulin resistance, promoted by different primary metabolic stressors, differential adaptive metabolic programming, and distinct metabolic profiles - but similar predisposition to deregulation of carbohydrate metabolism. In each of these in utero models, it is likely that nutrient sensing and fuel utilization have adapted to maintain optimal glucose levels during intrauterine stress. However such changes may become maladaptive during postnatal life, particularly when the nutrient/metabolic environment differs from the original metabolic stress. Thus, a key scientific and medical imperative is to identify distinct markers of metabolic aberrations, in the context of genetic and metabolic stress history, in order to develop tools for individualized approaches for “normalizing” maladaptive metabolic profiles in the context of a sedentary/hypercaloric contemporary life style.
Scope and aims: The MC-IOF project “Metabolic Plasticity” aims at establishing a model animal system for the study of the metabolic impact of nutritional, medicinal, hormonal and physical activity interventions that may be used therapeutically in states of compromised metabolism, especially those originating during early life. To accomplish this goal, studies aim at (a) the identification of measurable serum and tissue parameters (markers) that specifically characterize the progressive metabolic derangement (mainly carbohydrate intolerance) in distinct models of in utero metabolic stress and (b) the examination of the effects of nutritional, medical, and physical activity interventions on these markers and their association with changes in insulin resistance.
General hypothesis: Prenatal metabolic stress “programs” distinct, detectable adaptive responses which establish a specific pattern of fuel partitioning potentially maladaptive during postnatal life. Such responses may be reprogrammed by specific interventions in developmentally critical periods of life.
Specific hypotheses: (1) Serum and tissue metabolomes and components of the AMPK/mTOR sensor system are differentially altered under various types of intrauterine stress and may serve as markers of metabolic maladaptation. (2) Nutritional, caloric, exercise, medicinal or hormonal interventions, during developmentally critical periods in postnatal life can normalize these metabolic markers.
Specific objectives: (1a): To identify alterations in serum and tissue metabolomes, as well as cellular markers of metabolic aberrations in mice exposed to metabolic stress during prenatal life; (1b): To examine aspects of energy/fuel partitioning in animals exposed to metabolic stress during development; (2a): To determine whether interventions which can alter AMPK/mTOR sensor system responses can modulate phenotypes associated with intrauterine metabolic stress; (2b): To assess and validate the identified metabolic markers in in a human model of metabolic stress and altered insulin sensitivity.
Methods & Results: Experimental maternal undernutrition (UN) results in offspring predisposed to adiposity and progressive glucose intolerance. UN and control mice present with differential hepatic/adipose tissue growth patterns. To test the hypothesis that alterations in energy/nutrient sensing (e/Ns) may contribute to the development of increased adiposity and progressive glucose intolerance and may serve as relevant markers, we assessed the RNA/protein expression in liver and adipose tissue from male control and (UN) mice, at weaning (3 weeks) and early adult stages (10 weeks) of postnatal life. Microarray analysis of epididymal adipose tissue from 3 week old UN mice suggests that among AMPK/mTOR sensor system-related mRNAs, HNF-4a (increased by ~50%) and SREBP1 (decreased by ~40%) tended to be significantly altered, while FOXO1 mRNA was significantly reduced by 35%. In addition this analysis showed trends for reductions in PP2A-A (scaffolding subunit of protein phosphatase A), PP2A-B (regulatory subunit of PP2A which specifies substrate specificity), and PP2A-C (catalytic subunit of PP2A) isoform mRNA levels. PP2A is a major cellular phosphatase exerting inhibitory effects on the activities of multiple components of the AMPK/mTOR sensor system, such as, Akt, S6K, and mTOR; inhibition of mTOR can be antagonized by amino acids. At 10 weeks we assessed hepatic levels of phosphorylated (p-) components of the AMPK/mTOR sensor system and the protein levels of PP2A-A. Mice were fasted overnight and administered either leucine (C[LEU]; n=7, UN[LEU]; n=6) or vehicle (C[V]; n=9, UN[V]; n=7), by oral gavage, one hour before sacrifice and tissue collection. No significant PP2A-A, p-mTOR, p-Akt, or p-S6 differences were observed in 10 week adipose tissue. A significant reduction was observed in the hepatic PP2A-A levels in UN animals (by 24%, p<0.034 UN: n=13, C: n=16), while leucine administration had no significant effects on PP2A-A levels. In parallel, the correlation between p-mTOR and its autophagy-inhibiting effector, p-ULK1-Ser757 was disrupted in UN animals. To test the hypothesis that PP2A may modulate the sensitivity of the e/Ns system and the timing of cellular responses to nutrient withdrawal, we used siRNA-mediated PP2A-A knockdown in hepatoma cells and evaluated expression and signaling 72 hours later at baseline and/or after combined serum and glucose deprivation (SGD). PP2A-A knockdown (>50% decrease in protein) resulted in parallel decreases of PP2A-B and PP2A-C subunits. We observed significant condition dependent increases in the mTOR effector p-S6 at baseline and after 1h of SGD. Interestingly, PP2A-A knockdown also resulted in higher levels of the autophagy-inhibiting and mTOR-dependent p-ULK1-Ser757 at baseline and after 1h of SGD. While these differences were abolished after 4 hours of SGD, the levels of the autophagy marker LC3b were reduced at that time. Unexpectedly, p-Akt-Ser473 levels were substantially reduced by PP2A-A knockdown both at baseline and during SGD. We conclude that PP2A is a key controller of the AMPK/mTOR sensor system and alterations of PP2A-A levels distort hepatocyte e/Ns. We have also concluded that differential tissue allometric scaling in UN offspring coincides with a disturbed correlation between hepatic p-mTOR and its effector p-ULK1-Ser757 and inappropriately low PP2A-A, which signify e/Ns distortion. In a cohort of control and UN mice p-ULK1-Ser757 correlated linearly with body weight (r=-0.511 p<0.008) liver weight (LW; r=-0.66 p<0.0003) %LW (r=-0.593 p<0.002) and % epididymal fat weight (%EFW; r=0.434 p<0.03). Non-monotonic hormetic patterns describing correlations of birth weight with adult weight (inverted U-shaped) and PP2A-A (J-shaped), and of PP2A-A with the glucose/insulin ratio (U-shaped) were also observed. In UN mice we observed a striking U-shaped relationship between birth weight and %LW (r = 0.802 p<0.006). Furthermore, an inverted U-shaped pattern depicted the correlation between birth weight and adult hepatic p-ULK1-Ser757 in UN mice (r = 0.758 p<0.014). These findings suggest that developmental metabolic plasticity may impose non-linear hormetic correlation patterns linking birth weight with key elements of the e/Ns system; such hormetic patterns may drive differential tissue allometric scaling in UN mice and may provide an explanation for biological variability. “Predicted” fluctuations imposed by recognized non-monotonic patterns might allow normalization of variability through simple mathematical modelling.
The impact: Establishing experimental models and markers that will permit the assessment of the metabolic effects of lifestyle and medical interventions will be critical for enhancing our understanding of the pathophysiology of T2D, and for identifying individuals at increased risk. For our and similar models the recognition of non-monotonic/non-linear patterns regarding disease susceptibility or molecular features will have substantial impact on research design, as it will allow selection of subpopulations at risk through focusing on the most relevant fractions of hormetic curves and will enhance our ability to develop biomarkers for individualized diagnostics and follow up of preventive/therapeutic interventions for the T2D public health crisis.