4.1. Effect of CMO diet on maternal liver gene expression
This study showed that perinatal CMO intake affected expression of genes in the maternal liver related to energy and lipid metabolism compared to the control diet; changes included down-regulatation of genes such as Lcn2 (lipocalin 2) and Prkag1 (protein kinase AMP-activated non-catalytic subunit gamma). Reduction of Lcn2 (7.1 fold) and Prkag1 (1.5 fold), a subunit of 5' AMP-activated protein kinase, has been linked to inhibition of hepatic fatty acid oxidation and promotion of cholesterol synthesis, lipogenesis, and triglyceride synthesis (22, 23). Concentrations of lipids and other metabolites, however, were not confirmed by metabolomic or histological analysis of the liver.
When the concentration of hepatic lipids increases, the formation of very low-density lipoprotein (VLDL) particles is stimulated for packaging and circulation of triglycerides, reducing lipid accumulation in the liver (24). Indeed, up-regulation of Apob gene expression (2.2 fold) was observed in the dams’ liver; this gene provides instructions for making two versions of the apolipoprotein B protein, apolipoprotein B-48 and apolipoprotein B-100. Concentration of blood lipids, however, were not evaluated in this study. The time at which the dams’ livers were sampled (weaning) could also affect Apob gene expression, as increased plasma triglyceride and apoB-48 concentrations in dams at weaning have been observed in rats (25). Creb1 (cAMP-response element-binding protein), another up-regulated (1.6-fold) gene in dams fed CMO, has been shown to activate gluconeogenesis and fatty acid oxidation through increased expression of the nuclear hormone receptor peroxisome proliferator activated receptor gamma (PPAR-γ). However, PPAR-γ expression was not changed in the liver of dams fed the CMO diet (26).
Genes involved in ubiquitination and SUMOylation were also among differentially expressed transcripts in the liver of dams fed CMO compared to the control diet (Table 2). Post-translational modification of target protein substrates by ubiquitin-like proteins (Ubls) and SUMO proteins regulates cellular signalling in numerous processes such as metabolism, transcription, translation, vesicle transport and apoptosis. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s (27). The genes Ube2h, Ube2d3 and Ube2e3, expression of which was higher in dams fed CMO, encode members of the E2 ubiquitin-conjugating enzyme family, while Mul1 (mitochondrial ubiquitin protein ligase 1), Rnf220 (ring finger protein 220), Rnf111 and Trim56 (Tripartite Motif Containing 56) genes encode for E3 ubiquitin ligases (Table 2). E3 ubiquitin ligases accept ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfer the ubiquitin to targeted substrates. Among the different targets of E3 Ubs, Mul1 has been shown to play a role in the control of mitochondrial morphology and to have anti-apoptotic activity while Trim 56, and both Rnf111 and Rnf220, promote expression of a type I interferon IFN-beta in response to pathogens and enhance class I MHC mediated antigen processing and presentation. The expression of the SUMO specific peptidase 6 (Semp6) gene was increased in the liver of dams fed CMO compared to those fed the control diet; Senp6 regulates genome stability, cell division, autoimmune responses and ageing (28).
Diet effects on the liver ubiquitome and its role in the regulation of whole-body euglycemia and lipidemia have recently been reported in rats (29). More specifically, it was observed that ubiquitination of proteins is a key regulatory mechanism controlling fatty acid metabolism which may mediate the pathogenesis of fatty acid-associated diseases. Variations observed in this study on liver ubiquitination genes and genes involved in lipid metabolism combined with reduced liver weight suggest that CMO may have modified liver lipid metabolism towards increased energy absorption and utilization.
4.2. Effects of perinatal CMO consumption on offspring liver gene expression
In contrast to our hypothesis, the effects of perinatal diet on pups metabolic function at weaning may have led towards energy expenditure and not accumulation. However, it is also important to note that changes observed in pups liver gene expression at weaning may also be influenced by maternal diet, as pups close to weaning may consume small amounts of maternal diet that may fall into the cage. Pups from dams fed the CMO diet, for example, had increased expression of two genes involved in steroid metabolism at weaning. The expression of both Hsd3b5 (3 beta-hydroxysteroid dehydrogenase type 5) and Srd5a1 (3-oxo-5α-steroid 4-dehydrogenase 1) has been shown to negatively correlate with lipid accumulation in liver (30, 31). Although no changes in liver weight and other markers for liver lipid metabolism were found at weaning in pups from dams fed CMO, increased body growth (weight and length) may be correlated with changes in gene expression observed in the liver. Concentration of blood lipids, however, were not evaluated in this study.
Modulation of mitochondrial mass is also a major adaptive response to changes in energy balance, arising from decreases in oxygen or glucose availability, among other nutrient stresses (32). However, the effects of a maternal CMO diet on mitochondrial metabolism were not consistent, as the two pro-apoptotic genes differentially expressed in pups, Bcl-2 nineteen-kilo dalton interacting protein 3 (Bnip3) and Bcl-2-binding component 3 (Bbc3), were down-regulated and up-regulated respectively.
Other genes up-regulated in weaned pups from CMO-fed dams included those related to cytochrome P450s (CYP450) function. CYP450 monooxygenases are capable of catalyzing the metabolism of various endogenous and exogenous compounds, such as bile acids, fatty acids, retinoids, steroids, drugs and other xenobiotics. The effects of diet on CYP450 have been established and reviewed (33). An increase in protein-to-carbohydrate ratio in the diet, for example, was shown to increase products of steroid hormone metabolism contributing to the transcriptional regulation of drug-metabolizing genes P450s (34). Indeed, weaned pups from dams fed the CMO diet had increased intake of protein-to-carbohydrate ratio, as maternal milk had higher concentrations of protein compared to control fed dams (8).
By thirty days after weaning, pups from dams fed CMO compared to control diet, had no indication of expected changes in liver functionality, previously linked with increased visceral fat weight and serum leptin concentration (35). Analysis of the pups’ liver transcriptome by GSEA, however, showed that the expression of genes involved in the synthesis of epoxy (EET) and dihydroxyeicosatrienoic acids (DHET) and collagen metabolism were increased.
The EETs are signalling molecules formed within various types of cells by the metabolism of arachidonic acid by a specific subset of CYP450 enzymes. The EETs have been most studied in animal models where they show the ability to prevent arterial occlusive diseases such as heart attacks and brain strokes by their anti-hypertensive and anti-inflammatory effects on blood vessels (36). In the liver, collagens and proteoglycans play an intrinsic role in liver function in health and disease (37). The interaction of collagens and proteoglycans provide architectural elements for the liver with basement membrane or other duct architecture. They have mechanical roles like providing tensile strength and resilience, modulating diffusion and vascular flow, and regulating cell movement (37). Collagens and proteoglycans can also regulate signalling molecules such as growth factors, serving as ligands, storage depots and receptors, via multiple complex interactions between matrix proteins with other signal molecules or among different matrix components (38). However collagen deposition was not evaluated histologically in our study.
Traditionally, visceral obesity is strongly associated with insulin resistance, hypertension, dyslipidemia and systemic chronic low-grade inflammation, all of which play a pivotal role in the pathogenesis of atherosclerosis, thus increasing the risk of cardiovascular disease (39). However, as observed here, no expected changes on liver metabolism linked to the onset of metabolic diseases were observed in pups 30 days after weaning. This may be due to non-pathological residual effects of metabolic changes observed in early life or may be explained by mouse strain variations in response to diet-induced obesity (40).