Edited by: Katia Aquilano, Università degli Studi di Roma Tor Vergata, Italy
Reviewed by: Simone Carotti, Università Campus Bio-Medico, Italy; Wimal Pathmasiri, University of North Carolina at Chapel Hill, United States
This article was submitted to Nutrigenomics, a section of the journal Frontiers in Genetics
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Early life nutritional imbalances are risk factors for metabolic dysfunctions in adulthood, but the long term effects of perinatal exposure to high versus low protein diets are not completely understood. We exposed C57BL/6J offspring to a high protein/low carbohydrate (HP/LC) or low protein/high carbohydrate (LP/HC) diet during gestation and lactation, and measured metabolic phenotypes between birth and 10 months of age in male offspring. Perinatal HP/LC and LP/HC exposures resulted in a decreased ability to clear glucose in the offspring, with reduced baseline insulin and glucose concentrations in the LP/HC group and a reduced insulin response post-glucose challenge in the HP/LC group. The LP/HC diet group also showed reduced birth and weanling weights, whereas the HP/LC offspring displayed increased weanling weight with increased adiposity beyond 5 months of age. Gene expression profiling of hypothalamus and liver revealed alterations in diverse molecular pathways by both diets. Specifically, hypothalamic transcriptome and pathway analyses demonstrated perturbations of MAPK and hedgehog signaling, processes associated with neural restructuring and transmission, and phosphate metabolism by perinatal protein imbalances. Liver transcriptomics revealed changes in purine and phosphate metabolism, hedgehog signaling, and circadian rhythm pathways. Our results indicate maternal protein imbalances perturbing molecular pathways in central and peripheral metabolic tissues, thereby predisposing the male offspring to metabolic dysfunctions.
Metabolic syndrome and type 2 diabetes mellitus (T2DM) have reached epidemic proportions worldwide (
In the current study, we tested the hypothesis that intrauterine HP/LC and LP/HC diets would subsequently affect adult metabolic phenotypes and long-term gene expression levels in key metabolic sites. Using HP/LC or LP/HC dietary interventions during gestation and lactation, we followed male offspring until 10 months of age and assessed body weight, adiposity, insulin production, glucose clearance, and global gene expression changes in the hypothalamus and liver. We discovered that mice expressed differential programming by perinatal exposure to protein imbalances, and these changes persist into adulthood. Importantly, we found that gene expression changes brought about by perinatal nutritional perturbations revealed enrichment of genes involved in diverse pathways including those important for systemic energy homeostasis.
This study was approved by the UCLA Animal Research Committee and was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, United States).
The LP/HC diet (D02041002, Research Diets Inc., New Brunswick, NJ, United States) contains 9% protein, 4.4% fat, and 77% carbohydrates. The HP/LC diet (D02041001, Research Diets Inc., New Brunswick, NJ, United States) has 23% protein, 4.4% fat and 64% carbohydrate by weight. Standard chow diet (TD 7013, Harlan Teklad, Placentia, CA, United States), containing 18% protein, 6% fat, and 45% carbohydrate by weight, was used as the control diet. The diets were matched for total caloric content, making them isocaloric.
As most first litters did not survive, all the offspring were from second litters from dams 10 to 12 weeks old. C57BL/6 females were mated overnight with males between 10 and 16 weeks of age. Gestational day 0 (GD0) was determined by the detection of a vaginal plug in the morning. On GD8, pregnant females were placed on a LP/HC, HP/LC, or chow diet. Pups were weaned at 28 days of age into cages of four animals per cage, separated by sex and maternal dietary environment. A total of
Body weight of individual mice was measured on a scale, starting day 2 (birth weight) and thereafter monthly until sacrifice. Body composition was measured monthly until sacrifice by a rodent Nuclear Magnetic Resonance (NMR) scanner (Bruker Biospin, Billerica, MA, United States) that was standardized to an internal control provided by the manufacturer. Adiposity was determined as adipose tissue mass per unit (g) body weight.
Glucose tolerance tests were performed as previously described (
All figures demonstrate data displayed as means ± SEM, with the exception of insulin concentrations that were expressed as a ratio of 30 min/baseline values in Figure
Hypothalamus and liver were dissected between 10 AM and 12 PM from male mice at 10 months of age, placed immediately into liquid nitrogen, and stored at -80°C until RNA was extracted with the RNAeasy kit (Qiagen). A total of 40 males from three different
The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number
Birth weights, perinatal weights, and growth rates of pups exposed to different
In order to compare the impact of
A key metabolic syndrome indicator is glucose intolerance, which is a measure of baseline glucose, baseline insulin, and the ability to respond to an exogenous glucose load with adequate insulin production. We therefore carried out an Intraperitoneal Glucose Tolerance Test (IPGTT) at 9 months of age.
Early life exposure to LP/HC and HP/LC impaired the ability of offspring to clear glucose in adulthood. Both LP/HC and HP/LC mice had a significantly higher area under the curve (AUC) for blood glucose levels measured at 0.5, 1.0, and 1.5 h post glucose administration (Figure
Intraperitoneal Glucose Tolerance Test, baseline glucose, baseline insulin, and insulin response at 9 months of age for males LP/HC and HP/LC vs. control.
The metabolic phenotypic differences in adult offspring were accompanied by significant differences in gene expression in two metabolically relevant tissues, the liver and hypothalamus collected at final dissection conducted at 10 months of age. For the hypothalamus, we identified 1,441 and 843 differentially expressed genes (DEGs) for HP/LC and LP/HC groups, respectively, by Student’s
We queried the DEGs reaching
KEGG and GO pathway enrichment of differentially expressed genes in the hypothalamus, liver or both under different nutritional exposures in early life.
Pathways | Liver |
Hypothalamus |
||
---|---|---|---|---|
HP/LC | LP/HC | HP/LC | LP/HC | |
Phosphate metabolism | 4.10E-08 | 3.40E-04 | 1.50E-09∗ | 5.10E-04 |
Biopolymer modification | – | 5.80E-04 | 1.70E-07∗ | – |
Cellular localization of protein | – | 3.00E-04 | – | 7.00E-05 |
RNA processing | 3.70E-06 | 7.80E-04 | – | 7.10E-05 |
Nucleotide processing | – | – | 3.80E-05∗ | – |
Neural restructuring | – | – | 7.90E-05∗ | 3.00E-03 |
Protein transport | – | 2.00E-04 | 8.70E-06∗ | 5.00E-05 |
Cell cycle | 1.00E-05 | 2.10E-04 | 3.30E-03 | – |
Synaptic transmission | – | – | 1.00E-05∗ | – |
MAPK signaling pathway | – | – | 3.60E-05∗ | – |
Gap junction | – | – | 7.70E-05∗ | – |
Apoptosis | 8.70E-04 | – | 5.00E-04 | 1.50E-03 |
Purine metabolism | 5.50E-06∗ | – | – | – |
Hedgehog signaling pathway | – | 4.50E-03 | – | 5.20E-04 |
Colorectal cancer | 1.70E-05∗ | – | – | – |
Embryonic development | – | 3.60E-03 | 3.90E-03 | 1.60E-03 |
Oxidative phosphorylation | – | – | 3.80E-03 | 4.30E-05∗ |
Circadian rhythm related pathways | 6.30E-05 | 2.40E-04 | 1.20E-03 | – |
Long-term potentiation | – | – | 7.90E-05∗ | – |
Response to DNA damage stimulus | 1.80E-06∗ | – | – | – |
In both HP/LC and LP/HC conditions, shared KEGG/GO pathways within the hypothalamus include phosphate metabolism, oxidative phosphorylation, neural restructuring, apoptosis, and protein transport. Within the HP/LC condition, KEGG/GO pathways were specifically enriched for synaptic transmission, nucleotide processing, biopolymer modification, MAPK signaling pathway, circadian rhythm, long-term potentiation, and gap junction signaling in the hypothalamus. Within the LP/HC condition, hedgehog signaling was uniquely enriched (Supplementary Tables
The liver transcriptome pathway analysis showed differential gene expression for RNA processing, phosphate metabolism, cell cycle, and circadian rhythm in both nutritionally modified states. Purine metabolism, apoptosis, response to DNA damage stimulus and DNA repair within the liver were uniquely enriched within the HP/LC diets. Several pathways enriched among the genes altered by the LP/HC diet within the liver include biopolymer modification, cellular protein localization/transport, embryonic development, and hedgehog signaling (Supplementary Tables
Across the dietary groups and the two tissues, consistent pathways include circadian rhythm, phosphate metabolism, RNA processing, cell cycle/apoptosis, and embryonic development.
Our study showed that maternal diets with abnormal nutritional compositions such as HP/LC or a LP/HC diet led to developmental differences, which persisted into adulthood affecting glucose metabolism, body weight, adiposity and gene expression levels in the hypothalamus and liver. Male offspring from mothers exposed to LP/HC diet showed a significantly reduced birth weight, which persisted at weaning (day 28) and up to 10 months of age. Male offspring from the HP/LC group trended toward a higher birth weight, and had a significantly higher body weight at weaning and increased adiposity at 5 months. Additionally, both sets of offspring were found to have an impaired ability to clear glucose in adulthood. The IPGTT conducted in HP/LC and LP/HC litters resulted in a significantly higher AUC for both groups compared with control. Interestingly, the LP/HC diet presented offspring with reduced baseline insulin and glucose levels, whereas the HP/LC diet had a reduced insulin response 30 min after glucose exposure. This led us to investigate what genes and pathways might underlie these phenotypic differences in the offspring. We found diet- and/or tissue-specific genes and pathways (e.g., gap junction in hypothalamus and purine metabolism in liver for HP/LC, neuronal pathways for both diets in hypothalamus, hedgehog signaling for LP/HC in both tissues), as well as shared pathways across tissues and diets (e.g., cell cycle and circadian rhythm).
There is a well-cataloged history of studies exploring the effects of maternal diet alterations on diseases in adult offspring, specifically within the context of metabolic perturbations (
In our study, we essentially found a U-shaped curve in terms of the relationship between maternal protein intake and glucose tolerance in the offspring’s adult life. It is predicted that optimal levels for many essential nutrients follow a U-shaped curve: levels that are too low as well as too high result in detrimental health outcomes. Sufficient vitamin A, for instance, is essential in pregnancy to prevent diaphragmatic hernia in the fetus (
Our hypothalamic transcriptome and pathway analyses in the offspring indicate alterations in tissue gene programming, likely through the developmental consequences resulting from embryonic nutritional imbalances, that connects maternal diet with long-term physiological outcomes in offspring. Key hypothalamic pathways such as synaptic transmission, neural restructuring and neural development were particularly altered by the HP/LC maternal diet suggesting that these pathways may lead to abnormal neural circuitry in the hypothalamus, which plays a key role in energy balance, ultimately contributing to dysregulation of metabolic homeostasis including increased fat storage and glucose intolerance in adulthood. These findings corroborate a recent study where a high fat maternal diet led to significant alterations in hypothalamic gene expression, resulting in a disruption of regular hypothalamic circuitry through ECM and dopamine availability pathway perturbations (
In the liver, circadian rhythm and metabolic pathways such as phosphate metabolism and metabolic regulation were prominent, indicating that these pathways may be major effectors of metabolic syndrome outcomes due to altered maternal diets. Our findings that a number of circadian rhythm related pathways (Table
We also report the alteration of MAPK (for HP/LC) and hedgehog (for LP/HC) signaling pathways, both of which have been previously linked with the facilitation of insulin signaling. Disruption of insulin production or transduction of its signal is intimately associated with generation of metabolic syndrome (
We acknowledge that this is a discovery study and the findings provide the framework for future avenues of investigation. In particular, pathohistological and structural analysis of both the hypothalamus and liver tissues are necessary to show association between the highlighted pathways/genes and tissue pathology. Additionally, protein level assays and perturbation experiments are necessary to validate the functional and physiological outcomes of the potential causal genes highlighted by our current study. Moreover, it would be interesting to explore further the classification of adiposity and the relevant abundance of white versus brown adipose tissue. This would help characterize more precisely the effects of nutritional perturbation on adipogenesis. A potential limitation of this study is a lack of further validation of our Affymetrix data via RT-PCR, although the validity of microarrays has been highly supported in the literature (
Overall, the prevalence of metabolic disorders such as T2DM and metabolic syndrome in modern societies is growing and the etiology is clearly complex, involving diet, physical activity, and possibly endocrine disrupting chemicals in the environment (
LJM designed and conducted the experiments, researched the data, analyzed all the data, and drafted the manuscript. QM, SL, and CP analyzed the gene expression data. MB analyzed and summarized all the data, and wrote the manuscript. SX and WT conducted the experiments. JW researched and analyzed the data. SD developed experimental design and assisted in phenotypic experiments and interpretation. AJL developed the experimental design. XY supervised the data analysis and manuscript writing. All authors reviewed and edited the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: