Abstract
Background
Intrauterine growth restriction and low birth weight (LBW) have been widely reported as an independent risk factor for adult hypercholesterolaemia and increased hepatic cholesterol in a sex-specific manner. However, the specific impact of uteroplacental insufficiency (UPI), a leading cause of LBW in developed world, on hepatic cholesterol metabolism in later life, is ill defined and is clinically relevant in understanding later life liver metabolic health trajectories.
Methods
Hepatic cholesterol, transcriptome, cholesterol homoeostasis regulatory proteins, and antioxidant markers were studied in UPI-induced LBW and normal birth weight (NBW) male and female guinea pigs at 150 days.
Results
Hepatic free and total cholesterol were increased in LBW versus NBW males. Transcriptome analysis of LBW versus NBW livers revealed that “cholesterol metabolism” was an enriched pathway in LBW males but not in females. Microsomal triglyceride transfer protein and cytochrome P450 7A1 protein, involved in hepatic cholesterol efflux and catabolism, respectively, and catalase activity were decreased in LBW male livers. Superoxide dismutase activity was reduced in LBW males but increased in LBW females.
Conclusions
UPI environment is associated with a later life programed hepatic cholesterol accumulation via impaired cholesterol elimination in a sex-specific manner. These programmed alterations could underlie later life cholesterol-induced hepatic lipotoxicity in LBW male offspring.
Impact
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Low birth weight (LBW) is a risk factor for increased hepatic cholesterol.
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Uteroplacental insufficiency (UPI) resulting in LBW increased hepatic cholesterol content, altered hepatic expression of cholesterol metabolism-related genes in young adult guinea pigs.
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UPI-induced LBW was also associated with markers of a compromised hepatic cholesterol elimination process and failing antioxidant system in young adult guinea pigs.
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These changes, at the current age studied, were sex-specific, only being observed in LBW males and not in LBW females.
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These programmed alterations could lead to further hepatic damage and greater predisposition to liver diseases in UPI-induced LBW male offspring as they age.
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Introduction
Cholesterol is a critical biological molecule, acting as a precursor for the synthesis of steroid hormones, bile acids, and vitamin D, and also being critical as a central modulator of cell membrane proteins, receptor trafficking, signal transduction, and cell membrane fluidity.1 In mammals, the liver is the central organ regulating cholesterol homoeostasis through it actions in cholesterol uptake, export, conversion into bile acids, biosynthesis, and storage.2,3,4,5,6,7 When cholesterol homoeostasis is disrupted, large concentrations of cholesterol accumulate in the liver resulting in a lipotoxic state associated with oxidative stress and can culminate in nonalcoholic steatohepatitis (NASH).8,9,10,11,12,13,14
An increase in the consumption of dietary cholesterol and/or genetic susceptibility to hypercholesterolaemia can underlie impaired cholesterol homoeostasis, cholesterol accumulation, and a lipotoxic state in the liver.10,15,16 However, it is now becoming apparent that insults early in the life cycle, such as in prenatal life can also contribute, in a sex-specific manner, to aberrant cholesterol metabolism and hepatic cholesterol overload in adulthood. Animal studies have indicated that a wide variety of experimental in utero insult/stress situations (maternal protein undernutrition, dietary restriction, hypoxia, or prenatal nicotine) resulting in intrauterine growth restriction (IUGR)/Low Birth Weight (LBW) lead to increased serum and/or hepatic cholesterol in weaned and adult male rat offspring.17,18,19,20 Transcriptome analysis in the liver of prenatal maternal food restricted-adult IUGR rat offspring revealed that activated cholesterol biosynthesis and down-regulated bile acid biosynthesis pathways are main targets for intrauterine reprogramming.21 Studies have highlighted that IUGR models such as maternal protein undernutrition, bilateral uterine vessel ligation, or intrauterine stress induced via ‘sham’ surgery, yield different phenotypes and specific transcriptional patterns in organs such as placenta and kidney.22,23 Therefore, understanding in utero insult-specific mechanisms is key to identifying both conserved pathways and model-specific alterations which increase our understanding and applicability of how these modifications in organ systems and phenotype manifest. Collectively, this will allow for development of better-informed target interventions.
Observational human studies have reported that impaired in utero growth and the resulting LBW are associated with differentially increased serum concentrations of total and low-density lipoprotein (LDL) cholesterol in men and women in later life with sex difference by age groups in adults.24,25,26,27,28,29 Although, Uteroplacental Insufficiency (UPI) is the leading cause of IUGR/LBW in the developed world,30,31 investigations attempting to understand the specific impact of UPI-induced IUGR/LBW on later life hepatic cholesterol metabolism and cholesterolemia are currently limited to rat models.32,33 New reports have begun to examine the molecular pathways that may underly the association between abnormal foetal growth in utero and later life cholesterol metabolism dysregulation in humans, but to date they are limited to the analysis of cord blood plasma of IUGR at birth,34 with limited follow-up on hepatic cholesterol metabolism and associated pathways.
In the present study, we sought to unravel the hepatic metabolic pathways underlying the specific impact of UPI on hepatic cholesterol metabolism in young adult female and male LBW offspring. We have chosen to use the well-established pre-clinical animal model of UPI-induced IUGR/LBW in guinea pigs,35 especially given the close similarities between humans and guinea pigs with respect to uteroplacental development and function36 and later life hepatic and whole-body trafficking and processing of cholesterol.37 With the use of transcriptome analysis, we postulated that LBW offspring, in a sex-specific manner, would display increased hepatic cholesterol by young adulthood in association with alterations in key regulators of cholesterol metabolism.
Methods
Animals
All animal procedures were conducted in accordance with guidelines and standards of the Canadian Council on Animal Care. Animal Use Protocol (AUP-#2009-229) was approved and post approval monitoring conducted by the Western University Animal Care Committee. All investigators understood and followed the ethical principles outlined by Grundy,38 and study design was informed by ARRIVE guidelines.39
Time-mated pregnant Dunkin-Hartley guinea pigs (Charles River Laboratories, Wilmington, MA) were housed in 12 h light and dark cycles in a temperature (20 ± 2 °C) and humidity (30–40%)-controlled environment, with access to guinea pig chow (LabDiet diet 5025) and tap water ad libitum. All pregnant guinea pigs underwent uterine artery ablation35,40 at mid-gestation (~32 days, term ~69 days) and sows delivered spontaneously. At the end of the experimental pupping period, male and female pups were defined as normal birth weight (NBW) or low birth (LBW) as per previously reported criteria.40,41 At weaning (postnatal day 15), pups were housed individually in clear perplex containers and fed ad libitum a normal diet (TD.110239; Harlan Laboratories, Madison, WI) until young adulthood (postnatal day 150). Only one LBW and NBW pup of each sex, from a single litter, was used in order to minimize compounding effects of litter. At postnatal day (PND) 150, NBW (n = 8) and LBW (n = 8) offspring of each sex were fasted overnight, euthanized via CO2 inhalation42 in the morning (~10 a.m.) of the following day. The whole liver was removed and weighed immediately and the right lobe liver was frozen in liquid nitrogen and stored at −80 °C for later biochemical and molecular analyses.
Hepatic biochemical analysis
Hepatic triglycerides were measured using 50 mg of frozen liver and following the Triglyceride Colorimetric Assay Kit (Item No. 10010303; Cayman Chemical, Ann Arbor, MI). Liver content of total cholesterol, free cholesterol, and cholesteryl esters was measured as previously described.43,44 Enzymatic reagents for total cholesterol (WAKO Diagnostics: Cholesterol E (CHOD-DAOS method) #439-17501) and free cholesterol (WAKO Diagnostics: Free cholesterol (COD-DAOS) method #435-35801) were used as per the manufacturer’s instructions. Cholesteryl ester was determined as the difference between total cholesterol and free cholesterol.
RNA isolation
Total RNA for microarray analysis was isolated from a subcohort of five NBW and five LBW livers of each sex at the Genome Québec Innovation Centre (Montreal, QC, Canada) while independent validation of microarray data was performed using RNA extracted from all NBW and LBW animals of the experiment, including the microarray cohort. For the microarray analysis, 50 mg of frozen liver was homogenized in 1 mL of Trizol (Invitrogen, Burlington, ON, Canada). The homogenate was treated with 100 μL of gDNA eliminator solution and then shaken vigorously and maintained at room temperature for 2–3 min. The homogenate was centrifugated at 12,000g for 15 min at 4 °C. RNA in the aqueous phase was then extracted using the RNeasy Plus Universal Mini Kit (Qiagen, Toronto, ON, Canada). Total RNA for validation of microarray data was isolated from frozen ground liver (100 mg) using Trizol reagent and the Purelink RNA Mini Kit according to the manufacturer’s instructions (Invitrogen). The quantity and quality of extracted RNA was assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. RNA samples with RNA Integrity Number (RIN) above 7 were considered of high integrity and considered for microarray and quantitative reverse transcription PCR (RT-qPCR) analyses.45
RNA labelling, microarray hybridization, scanning, and processing
A Whole-Transcript Expression Analysis Gene Titan was conducted (Genome Québec Innovation Centre). Sense-strand cDNA was synthesized from 100 ng of total RNA, and fragmentation and labelling were performed to produce ss-cDNA with the GeneChip® WT Terminal Labeling Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). After fragmentation and labelling, 2.1 μg cDNA was hybridized onto Guinea Pig Gene 1.1 ST Array Plate (Thermo Fisher Scientific) and processed on the GeneTitan® Instrument (Thermo Fisher Scientific) for Hyb-Wash-Scan automated workflow. The microarray scanned images were imported into the Transcriptome Analysis Console (TAC) (version 4.0) (Life Technologies, Carlsbad, CA) and the raw microarray data (.CEL files) were normalized using the Robust Multiple-Array Averaging (RMA) method. Differentially expressed genes were identified using the TAC Expression (Gene) analysis method. Microarray data have been deposited in the National Center for Biotechnology information Gene Expression Omnibus (GEO; accession number GSE161124).
Quantitative reverse transcription PCR
Independent validation of microarray data was performed by examining levels of selected differentially expressed transcripts using RT-qPCR as previously described.46 Detailed information for primer sequences is provided in Supplementary Table 1. Gene expression analysis was performed with the CFX Maestro software (Bio-rad, Mississauga, ON, Canada) using the 2−∆∆Ct method and GAPDH and beta actin as reference genes. GAPDH and beta actin were classified as acceptable reference genes by GeNorm algorithm in the CFX Maestro software with a calculated expression stability value (M) of 0.57 for both genes. Threshold (Ct) values were also consistent between the NBW and LBW groups.
Immunoblot analysis
Proteins were extracted from frozen ground liver (50 mg) in radioimmunoprecipitation assay buffer as previously described.46 Equal amounts of total proteins (20 μg) were separated on a 7.5 or 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were then blocked with 5% non-fat milk in 0.1% TBS-Tween-20 and probed with primary and secondary antibodies (Supplementary Table 2). The chemiluminescence signal was captured with the ChemiDoc MP Imaging System (Bio-Rad), and protein band densitometry was determined using the Image Lab software (Bio-Rad). Ponceau staining was used as the loading control, as previously published.47,48
SOD, CAT, GSH, and GSSG assays
Superoxide dismutase (SOD; kit no. 706002, Cayman Chemical) and catalase (CAT; kit no. 707002, Cayman Chemical) enzyme activities, reduced (GSH), and oxidized (GSSG) glutathione (kit no. 703002, Cayman Chemical) in livers were measured following the kit instructions.
Statistical analysis
Non-microarray data
A Shapiro–Wilks test was used to determine if data were normally distributed. The few of the data sets that did not pass the normality test and comparing four experimental groups (birth weight, abdominal circumference, CRL, naso-anal length and Lee index at birth, liver:body weight ratio and free cholesterol at PND 150) were thus Box−Cox-transformed before ANOVA. A two-way ANOVA was used to determine the main effect of birth weight, sex, and possible interactions, followed by Bonferroni post hoc test using GraphPad 8 (GraphPad Software, San Diego, CA). When comparing NBW and LBW only, a Student’s t-test was used for normally distributed and a Mann–Whitney was performed on non-normally distributed data. Data are presented as mean ± SEM and a probability of 5% (p < 0.05) was considered significant for all analyses.
Microarray data
Only genes that met the criteria of |fold change|≥2 and p value <0.05 were defined as differentially expressed. To explore biological processes and pathways associated with the differentially expressed genes, we performed gene ontology (GO) biological process term enrichment (adjusted p < 0.05) using human as target organism and the g:GOSt functional profiling in g:Profiler web server (https://biit.cs.ut.ee/gprofiler/gost).
Results
Phenotypic traits
By using diathermy partial ablation of branches of the uterine artery, UPI was induced to generate LBW, as described previously.35 Those pups that at birth were <85 g, were classified as LBW and as a group weighed less than sex-matched NBW pups (p < 0.0001; Table 1). At birth, crown–rump length (CRL) tended to decrease (p < 0.090) and biparietal diameter (BPD) as well as body weight over naso-anal length (BW:length) decreased (p < 0.01) in LBW offspring (Table 1). In addition, at birth, Lee index (body weight × 0.33:naso-anal length), a measure of fatness,49 decreased in both male and female LBW offspring (p < 0.01; Table 1). At the time of tissue collection (PND 150; Table 1), LBW and NBW offspring had similar mean body and absolute and relative liver weights within each sex group. However, at this age, females displayed overall lower body, absolute and relative liver weights than males, independent of the birth weight (p < 0.05; Table 1). Male offspring overall exhibited higher hepatic free cholesterol and total cholesterol than females (p < 0.01; Table 1). Interestingly, hepatic-free cholesterol and total cholesterol contents were increased in LBW males compared to NBW males (p < 0.01; Table 1). No significant difference was observed for hepatic-free cholesterol and total cholesterol content in NBW or LBW females (Table 1). Birth weight or sex at PND 150 had no impact upon hepatic cholesteryl ester and triglyceride contents (Table 1).
Hepatic transcriptome
Analysis of whole hepatic transcriptome results identified that between LBW and NBW offspring livers, 51 (29 up-regulated and 22 down-regulated), and 31 (8 up-regulated and 23 down-regulated) genes were differentially expressed (|fold change| ≥ 2 and p value <0.05) in males and females respectively (Fig. 1 and Supplementary Tables 3 and 4). The Venn diagram (Fig. 1a), the volcano plots (Fig. 1b, c), and 2D hierarchical clustering chart/heat maps (Fig. 2a, b) indicate the degree of separation among the LBW and NBW groups in males and females. Only two genes encoding thioredoxin pseudogene and small nucleolar RNA SNORA2/SNORA34 family transcripts were differentially expressed between LBW and NBW in both male and female offspring (Fig. 1a).
Functional analysis of the hepatic transcriptomic profile and validation of microarray data
In the biological process analysis of the differentially expressed genes in LBW versus NBW males, 18 enriched GO biological processes were observed, with positive regulation of hepatic fatty acid metabolism process, lipid transport, lipid localization, regulation of fatty acid biosynthetic process and positive regulation of lipid metabolic process being the five top ranked processes (Fig. 3a and Table 2). No significant changes to GO biological processes were observed in LBW females (Fig. 3b). The KEGG functional enrichment analysis identified that cholesterol metabolism was a significantly enriched pathway in LBW males (adjusted p < 0.05, Fig. 3a). Specifically, in LBW males, the reverse cholesterol transport-related genes (Apoa1 and Angplt4) were up-regulated while Ldlr gene associated with the internalizing of circulating LDL cholesterol was down-regulated (Table 2 and Supplementary Table 3).
The PPAR signalling pathway was also an up-regulated pathway in LBW versus NBW offspring (adjusted p < 0.05, Fig. 3a). This latter pathway included Adipoq in addition to Apoa1 and Angplt4 (Table 2 and Supplementary Table 3). FoxO signalling pathway was the only significantly enriched pathway for differentially expressed genes in LBW females (adjusted p < 0.05, Fig. 3B). This pathway included down-regulated Bcl6 and Gadd45g (Table 2 and Supplementary Table 4).
Genes involved in cholesterol metabolism, PPAR and FoxO signalling pathways and other differentially expressed genes not related to these pathways were selected to be validated by RT-qPCR independent RNA samples (Fig. 4). Apoa1 gene was increased 4.76-fold (p = 0.037) in LBW relative to NBW males, similar to the fold change in the microarray data of 2.92-fold (p = 0.037). Angplt4 was also confirmed to be increased by 3.7 (p = 0.023) similar to microarray fold change of 2.84 and 1.2 (p = 0.011). In LBW versus NBW males, Ldlr and Gstt2 genes were also confirmed to be decreased by −2.16 and −3.05-fold (p = 0.008 and 0.020) and similar to microarray fold changes of −2.39 and −2.44, respectively (p = 0.021 and 0.041). Inhba, which was −2.82-fold lower in the microarray data (p = 0.036), was relatively lower in the LBW male livers by 2.89-fold (p = 0.061). Although not significant, Anxa1, which was 2.18-fold higher in the microarray data (p = 0.015), was relatively higher in the LBW male livers by 1.72-fold (p = 0.721). Lbp was significantly lower by −2.28-fold in the LBW female livers in the validation cohort (p = 0.038), similar to the −2.96-fold decreased in the LBW microarray female cohort (p = 0.026). Lastly, while down-regulated in LBW females in the microarray data, Lcn2 tended to decrease in the validation cohort (p = 0.105).
Hepatic content of proteins related to cholesterol metabolism
Selected proteins were studied on a priori based on their known biological role in hepatic cholesterol metabolism. The levels of hepatic proteins involved in cholesterol uptake (Ldlr, Apoe), biosynthesis (Srebp2), catabolism (Cyp7a1), efflux into bile acids (Abcg8), and export to blood (Fas, Acc, Abca1, and Mtp) were determined in LBW and NBW livers (Fig. 5). Female livers displayed significantly higher levels of Ldlr and Srebp2 protein than males (p < 0.01). Neither birth weight nor sex impacted Apoe protein. Females displayed lower Mtp and Acc protein levels than males (p < 0.01) and Fas protein levels were not affected by birth weight nor sex. Cyp7a1 and Mtp protein levels were both reduced in male LBW compared to male NBW livers.
Hepatic antioxidant systems
As elevated hepatic cholesterol is associated with oxidative stress,9,10 we quantified markers of this latter. The protein levels and activities of antioxidant enzymes SOD and CAT, as well as GSH and GSSG concentrations were determined in LBW and NBW livers. Sex had a significant effect on SOD1 protein level, which were lower in females than in males (p < 0.0001; Fig. 6a). While hepatic CAT protein was not impacted by birth weight nor sex, hepatic CAT activity was lower in male LBW (p < 0.05) (Fig. 6a, b). SOD activity was decreased in male LBW but increased in female LBW (Fig. 6c). GSH activity was not significantly affected by birth weight or sex (Fig. 6d). While the concentration of GSSG was significantly reduced in male LBW compared to male NBW (p < 0.05; Fig. 6e), the ratio of GSH:GSSG was not significantly impacted by birth weight or sex (Fig. 6f).
Discussion
An adverse environment during in utero life has been associated with gene reprogramming and modification of organ functions that can persist throughout the entire lifespan and are associated with an increased risk of developing the metabolic syndrome.50 Using a pre-clinical guinea pig animal model of UPI, we demonstrated that LBW is associated with increased hepatic cholesterol content and aberrant expression of cholesterol metabolism-related signalling pathway genes in young adulthood (PND150) liver. These changes occur in conjunction with markers of a compromised hepatic cholesterol elimination process and indications of antioxidant stress in young adults. Interestingly, these changes, at the current age studied, were sex-specific, only being observed in LBW males.
The impact of an adverse in utero environment on cholesterol metabolism has been mostly studied using mouse and rat models of IUGR arising from maternal protein undernutrition, in utero dietary restriction, prenatal hypoxia, or nicotine exposure.17,18,19,20 These different species and intrauterine insults collectively lead to elevated serum or hepatic cholesterol levels at weaning or in the adulthood in male offspring, similar to what was observed in this current study. The specific aetiology of the IUGR insult is of critical importance in determining the adult metabolic outcomes and phenotype22,23 and hence in the current study, UPI was investigated as it is the most common in utero insult associated with IUGR and LBW in developed world.51,52 Moreover, the guinea pig was utilized given its greater similarities with human pregnancy and outcomes, including a relatively long gestation, haemomonochorial placenta, luteo‐placental shift in hormone production, foetal development of metabolic tissues, and precocial offspring.36 The reported outcomes here are similar to the observed increased in blood total cholesterol at 15 weeks of age observed in UPI-induced IUGR male rat offspring.32 Additionally, the observed increase in hepatic cholesterol without increased hepatic triglycerides in male LBW offspring is also in alignment with other species studies that report IUGR-induced alterations in the cholesterol metabolizing pathway, without changes in hepatic fatty acid metabolism.17,33 The current work highlights not only that the UPI environment is associated with programming of later life hepatic cholesterol metabolic dysfunction, but also validates the experimental in utero insults/stress situations used in other species. Collectively, this provides external validation for the central nature of this pathway and later life cholesterol metabolic dysregulation across species when subjected to in utero environments associated with LBW outcomes.
The present study also highlights that cholesterol metabolism and PPAR signalling pathways are impacted and associated genes are differentially expressed in LBW versus NBW male offspring, but not in females. Hepatic Apoa1, Angplt4, and Adipoq genes, components of both cholesterol metabolism and PPAR signalling pathways, were up-regulated exclusively in LBW males. Apoa1 is the main apolipoprotein of high-density lipoprotein (HDL), playing a key role in regulating lipid transport and in the process of reverse cholesterol transport acting through promotion of the efflux of excess cholesterol from peripheral tissues and returning it to the liver for biliary excretion.53 Hepatic ApoA-1 gene expression is up-regulated in liver disease states,54,55 and ApoA-1 protein among other liver-specific proteins in extracellular vesicles, has been suggested to potentially serve as a specific biomarker for hepatotoxicity in drug- and alcohol-mediated hepatic injury.56 Additionally, Angptl4 and Adipoq, like ApoA-1, collectively serve a number of important roles in cholesterol metabolism. Angptl4 is present in HDLs physically protecting HDLs from endothelial lipase hydrolysis,57 and upregulates cholesterol synthesis in liver secondary to inhibition of lipoprotein lipase- and hepatic lipase-dependent hepatic cholesterol uptake.58 In addition, Adipoq accelerates reverse cholesterol transport by increasing HDL assembly through enhanced ApoA-1 synthesis in the liver.59 Conversely, the decreased Ldlr gene expression and unaltered Ldlr protein in LBW males may reflect an unaltered LDL cholesterol uptake into these livers. In agreement with this assumption, it is well established that overaccumulation of cholesterol in the liver supresses Ldlr gene transcription and accelerates its mRNA decay.60 Finally, the intersection of cholesterol and PPAR pathways is of note given the relationship between PPARs in the regulation of bile acid and cholesterol homoeostasis.61 Therefore, the current changes in gene expression and the previously observed unchanged serum cholesterol levels in this model62 collectively emphasize a potential increased HDL assembly and/or reverse cholesterol transport. This likely occurs in conjunction with an unaltered LDL uptake in LBW male liver, which may underly the higher hepatic cholesterol content observed, though this remain to be more thoroughly investigated.
Although unaltered at transcriptional level, hepatic Cyp7a1 and Mtp were reduced at the protein level in LBW males. The Cyp7a1 enzyme catalyses the initial step in cholesterol catabolism and bile acid synthesis and decreased Cyp7a1 gene expression and protein levels have been reported in conjunction with increased hepatic cholesterol in adult IUGR male rats from protein-restricted mothers.17 Further, Cyp7a1-deficient mice are observed to have elevated hepatic and serum cholesterol and decreased total bile acids,63 supporting the concept that Cyp7A1 acts in a similar manner in guinea pigs and rats and that alterations in its level/activity impact hepatic cholesterol content. It is of further interest to note that the human CYP7A1 mutation results in substantial cholesterol accumulation in the liver as well as decreased classic bile acid synthesis.64 Our current findings are in line with the concept that Cypa7a1 is critical in the control of hepatic cholesterol homoeostasis and provide evidence that Cyp7a1-induced cholesterol catabolism appears sensitive to changes in the intrauterine environment, specifically those associated with UPI such as hypoxia and altered nutrient supply.17,19 The parallel decrease in Mtp protein is of great interest considering that Mtp catalyses the assembly of cholesterol, triglycerides, and apolipoprotein B to VLDL for their export outside of the liver.65 Therefore, this impairment in hepatic cholesterol catabolism/efflux-related proteins could reflect a lower elimination rate of hepatic cholesterol, programmed by an adverse in utero environment, which culminates in increased cholesterol content in LBW males born from pregnancies complicated by UPI.
Antioxidant enzyme defense and non-enzymatic antioxidant defense systems are critical in reducing oxidative stress and maintaining redox homoeostasis within liver.66 SOD reduces the radical superoxide to form hydrogen peroxide and oxygen67 and CAT catalytically decomposes hydrogen peroxide into water and oxygen.68 At the same time, glutathione peroxidase (GPx) also reduces hydrogen peroxide to water while converting GSH to GSSG.69 During oxidative stress there is decrease in levels of GSH and increase in levels of GSSG and thus GSH/GSSG ratio decreases.70 Liver oxidative injury and abnormal activities of hepatic antioxidant enzymes are reported in male neonates from IUGR pregnancies.71,72 In the current report, UPI-induced LBW male offspring displayed a significantly depressed CAT activity, and a reduced total SOD activity, a result not observed in the LBW female livers. The ratio of GSH to GSSG was however not reduced in the livers of LBW males. Collectively, these data suggest that whereas female offspring appear to have functional postnatal hepatic oxidative stress recovery mechanisms, the in utero defects in male offspring appear to persist, and this could manifest with the altered hepatic cholesterol metabolic pathways observed. Indeed, in adult animal cholesterol feeding studies, elevated hepatic cholesterol is associated with oxidative stress.9,10 It is then possible that alterations in the functional postnatal hepatic oxidative stress of male LBW start at the level of SOD and CAT enzymes and may later extend to the glutathione redox couple GSH/GSSG system, as previously highlighted.70 This notion maybe supported by the observed reduction in the expression of inhba and Gstt2 genes, involved in hepatocyte regeneration and antioxidant system, respectively.73,74 Therefore, our current observations point to a lower antioxidant capacity in young adult LBW male offspring born from UPI pregnancies, which may promote the development of liver diseases in later life, especially when challenged with an elevated cholesterol environment.
In the current study, we observed lower body and liver weights at young adulthood in females than males, independent of the birth weight. Previous studies have also reported a sexual dimorphism in guinea pig with males consistently larger than females in skeletal measurements and body weight.75,76 It has been proposed that rapid and early growth in males leads to male-biased sexual dimorphism in these cases. In conjunction with these growth differences, we report sexual dimorphism in hepatic Ldlr, Srebp2, Mtp, Acc, and SOD1 proteins as well as liver cholesterol content, irrespective of birth weight, but also sex-specific programming of Apoa1, Angplt4, and ldlr genes and Cyp7a1 and Mtp proteins in male LBW offspring. Epidemiologic studies have demonstrated that LBW predisposes to adult onset hypercholesterolaemia in men,26,27,28 and women29 with sex difference by age groups in adults.24 In the current study, male offspring displayed higher hepatic free cholesterol and total cholesterol than females, despite higher protein abundance of Ldlr and Srebp2 and reduced Mtp and Acc proteins in female livers. We speculate that oestrogens levels may be protective for cholesterol overaccumulation within livers of female guinea pigs, given that physiological levels of oestrogen increased CYP7A1 activity along with small transient increases in bile acid production in hepatocytes.77 These results also indicate that protective compensatory mechanisms of elevated Ldlr and Srebp2 protein in males and reduced Mtp and Acc protein in females occurs.
A limitation of our study is that the concentrations of sex steroids in the serum as well as the steroid receptors in the liver were not measured. Cholesterol is the precursor for endogenous sex steroid biosynthesis.1 Furthermore, testosterone replacement therapy enhances liver cholesterol uptake, suppressing cholesterol removal, and promoting cholesterol storage.78 Oestrogen together with oestrogen receptor-α also plays a role in preventing liver malfunctioning and reducing liver damage.79 Thus, a role of serum sex steroids and downstream signalling on the observed hepatic outcomes cannot be ruled out. Future work should also examine serum concentrations of triglycerides and cholesterol types in order to better characterize uptake/export of these lipids from livers of LBW animals.
In conclusion, the present study demonstrates that LBW occurring as a result of UPI results in increased hepatic cholesterol content, likely through compromised hepatic cholesterol elimination in young male, but not female, guinea pigs. The mechanisms underlying this potential programing effect and continued postnatal presentation of the defect are yet to be fully delineated. Certainly UPI is associated with hypoxia80 and foetal hypoxia is associated with promoted oxygenated blood flow to the heart and reduced umbilical blood supply to foetal liver, an adaption which is understood to reprogramme liver carbohydrate and lipid metabolism in utero81,82,83,84 likely through altered epigenetic regulation of hepatic metabolism.17 Furthermore, the observed disrupted hepatic cholesterol metabolism may contribute to permanent alterations in hepatic oxidative stress defense system, ultimately leading to further hepatic damage and greater predisposition to liver diseases in UPI-induced LBW male offspring as they age.
References
Lin, C. et al. Impact of cholesterol on disease progression. Biomedicine 5, 1–7 (2015).
Trapani, L., Segatto, M. & Pallottini, V. Regulation and deregulation of cholesterol homeostasis: the liver as a metabolic “power station”. World J. Hepatol. 4, 184–190 (2012).
Sharpe, L. J. & Brown, A. J. Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). J. Biol. Chem. 288, 18707–18715 (2013).
Feingold, K. R. in Diagnosis and Treatment of Diseases of Lipid and Lipoprotein Metabolism in Adults and Children (eds. Feingold, K. R. et al.) (MDText.com, Inc., 2000).
Zinkhan, E. K. et al. Intrauterine growth restriction combined with a maternal high-fat diet increases hepatic cholesterol and low-density lipoprotein receptor activity in rats. Physiol. Rep. 4, 1–13 (2016).
Kosters, A. et al. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice. J. Lipid Res. 47, 1959–1966 (2006).
Pandak, W. M. et al. Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis. Am. J. Physiol. Gastrointest. Liver Physiol. 281, 878–889 (2001).
Domínguez-pérez, M. et al. Hepatocyte growth factor reduces free cholesterol-mediated lipotoxicity in primary hepatocytes by countering oxidative stress. Oxid. Med. Cell Longev. 2016, 1–8 (2016).
Nuño-lámbarri, N. et al. Liver cholesterol overload aggravates obstructive cholestasis by inducing oxidative stress and premature death in mice. Oxid. Med. Cell Longev. 2016, 1–13 (2016).
Pérez, M. D. et al. Cholesterol burden in the liver induces mitochondrial dynamic changes and resistance to apoptosis. Cell Physiol. 234, 7213–7223 (2019).
López-islas, A. et al. Cholesterol enhances the toxic effect of ethanol and acetaldehyde in primary mouse hepatocytes. Oxid. Med. Cell Longev. 2016, 1–9 (2016).
López-Reyes, A. et al. Cholesterol overload in hepatocytes affects nicotinamide adenine dinucleotide phosphate oxidase (NADPH) activity abrogating hepatocyte growth factor (HGF) induced cellular protection. Gac. Med. Mex. 151, 456–464 (2015).
Ioannou, G. N., Haigh, W. G., Thorning, D. & Savard, C. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis. J. Lipid Res. 54, 1326–1334 (2013).
Ioannou, G. et al. Cholesterol crystals in hepatocyte lipid droplets are strongly associated with human nonalcoholic steatohepatitis. Hepatol. Commun. 3, 776–791 (2019).
Paththinige, C. S., Sirisena, N. D. & Dissanayake, V. Genetic determinants of inherited susceptibility to hypercholesterolemia—a comprehensive literature review. Lipids Health Dis. 16, 1–22 (2017).
Bellanti, F. et al. Redox biology synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression. Redox Biol. 15, 86–96 (2018).
Sohi, G., Marchand, K., Revesz, A., Arany, E. & Hardy, D. B. Maternal protein restriction elevates cholesterol in adult rat offspring due to repressive changes in histone modifications at the cholesterol 7alpha-hydroxylase promoter. Mol. Endocrinol. 25, 785–798 (2011).
Zhou, J. et al. Two intrauterine programming mechanisms of adult hypercholesterolemia induced by prenatal nicotine exposure in male offspring rats. FASEB J. 33, 1110–1123 (2019).
Rueda-clausen, C. F. et al. Hypoxia-induced intrauterine growth restriction increases the susceptibility of rats to high-fat diet-induced metabolic syndrome. Diabetes 60, 507–516 (2011).
Choi, G. Y. et al. Gender-specific programmed hepatic lipid dysregulation in intrauterine growth-restricted offspring. Am. J. Obstet. Gynecol. 196, 1–7 (2007).
Lecoutre, S. et al. Transcription profiling in the liver of undernourished male rat offspring reveals altered lipid metabolism pathways and predisposition to hepatic steatosis. Am. J. Physiol. Endocrinol. Metab. 317, E1094–E1107 (2019).
Nüsken, K. D. et al. Fetal programming of gene expression in growth-restricted rats depends on the cause of low birth weight. Endocrinology 152, 1327–1335 (2011).
Nüsken, E. et al. Altered molecular signatures during kidney development after intrauterine growth restriction of different origins. J. Mol. Med. 98, 395–407 (2020).
Chen, L. et al. Relationship between birth weight and total cholesterol concentration in adulthood: a meta-analysis. J. Chin. Med. Assoc. 80, 44–49 (2017).
Barker, D. J., Martyn, C. N., Osmond, C., Hales, C. N. & Fall, C. H. Growth in utero and serum cholesterol concentrations in adult life. BMJ 307, 1524–1527 (1993).
Ziegler, B. et al. Inverse association between birth weight, birth length and serum total cholesterol in adulthood. Scand. Cardiovasc. J. 34, 584–588 (2000).
Cooper, R. & Power, C. Sex differences in the associations between birthweight and lipid levels in middle-age: findings from the 1958 British birth cohort. Atherosclerosis 200, 141–149 (2008).
Davies, A. A., Smith, G. D., Ben-Shlomo, Y. & Litchfield, P. Low birth weight is associated with higher adult total cholesterol concentration in men: findings from an occupational cohort of 25 843 employees. Circulation 110, 1258–1262 (2004).
Miura, K. et al. Birth weight, childhood growth, and cardiovascular disease risk factors in Japanese aged 20 years. Am. J. Epidemiol. 153, 783–789 (2001).
Henriksen, T. & Clausen, T. The fetal origins hypothesis: placental insufficiency and inheritance versus maternal malnutrition in well-nourished populations. Acta Obstet. Gynecol. Scand. 81, 112–114 (2002).
Ciurea, E. L. Morphological survey of placenta in trombophilia related hypoperfusion of maternal-fetal blood flow. Curr. Heal Sci. J. 44, 85–91 (2018).
Nüsken, K., Dötsch, J., Rauh, M., Rascher, W. & Schneider, H. Uteroplacental insufficiency after bilateral uterine artery ligation in the rat: impact on postnatal glucose and lipid metabolism and evidence for metabolic programming of the offspring by sham operation. Endocrinology 149, 1056–1063 (2008).
Zinkhan, E. K. et al. Combination of intrauterine growth restriction and a high-fat diet impairs cholesterol elimination in rats. Pediatr. Res. 76, 432–440 (2014).
Miranda, J. et al. Metabolic profiling and targeted lipidomics reveals a disturbed lipid profile in mothers and fetuses with intrauterine growth restriction. Sci. Rep. 8, 1–14 (2018).
Turner, A. J. & Trudinger, B. J. A modification of the uterine artery restriction technique in the guinea pig fetus produces asymmetrical ultrasound growth. Placenta 30, 236–240 (2009).
Morrison, J. L. et al. Guinea pig models for translation of the developmental origins of health and disease hypothesis into the clinic. J. Physiol. 23, 5535–5569 (2018).
Fernandez, M. L. & Volek, J. S. Guinea pigs: a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation. Nutr. Metab. 3, 1–6 (2006).
Grundy, D. Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology. J. Physiol. 593, 2547–2549 (2015).
Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: The ARRIVE Guidelines for Reporting Animal Research. PLoS Biol. 8, e1000412 (2010).
Sarr, O., Thompson, J. A., Zhao, L., Lee, T. Y. & Regnault, T. R. H. Low birth weight male guinea pig offspring display increased visceral adiposity in early adulthood. PLoS ONE 9, 1–13 (2014).
Thompson, J. A., Gros, R., Richardson, B. S., Piorkowska, K. & Regnault, T. R. H. Central stiffening in adulthood linked to aberrant aortic remodeling under suboptimal intrauterine conditions. AJP Regul. Integr. Comp. Physiol. 301, R1731–R1737 (2011).
Greulich, S. et al. Secretory products of guinea pig epicardial fat induce insulin resistance and impair primary adult rat cardiomyocyte function. J. Cell Mol. Med. 15, 2399–2410 (2011).
Samsoondar, J. P. et al. Prevention of diet-induced metabolic dysregulation, inflammation, and atherosclerosis in Ldlr-/- mice by treatment with the ATP-citrate lyase inhibitor bempedoic acid. Arter. Thromb. Vasc. Biol. 37, 647–656 (2017).
Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).
Choi, S., Ray, H. E., Lai, S. H., Alwood, J. S. & Globus, R. K. Preservation of multiple mammalian tissues to maximize science return from ground based and spaceflight experiments. PLoS ONE 11, 1–14 (2016).
Sarr, O. et al. Western diet consumption through early life induces microvesicular hepatic steatosis in association with an altered metabolome in low birth weight Guinea pigs. J. Nutr. Biochem. 67, 219–233 (2019).
Lojpur, T. et al. Δ9-Tetrahydrocannabinol leads to endoplasmic reticulum stress and mitochondrial dysfunction in human BeWo trophoblasts. Reprod. Toxicol. 87, 21–31 (2019).
Sander, H., Wallace, S., Plouse, R., Tiwari, S. & Gomes, A. V. Ponceau S waste: Ponceau S staining for total protein normalization. Anal. Biochem. 575, 44–53 (2019).
Rogers, P. & Webb, G. P. Estimation of body fat in normal and obese mice. Br. J. Nutr. 43, 83–86 (1980).
Gluckman, P., Hanson, M., Phil, D., Cooper, C. & Thornburg, K. Effect of in utero and early-life conditions on adult health and disease. N. Engl. J. Med. 359, 61–73 (2014).
Brown, L. D. & Hay, W. W. Impact of placental insufficiency on fetal skeletal muscle growth. Mol. Cell Endocrinol. 435, 69–77 (2017).
Kinzler, W. L. & Vintzileos, A. M. Fetal growth restriction: a modern approach. Curr. Opin. Obstet. Gynecol. 2, 125–131 (2008).
Lewis, G. F. & Rader, D. J. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ. Res. 96, 1221–1232 (2005).
Panduro, A., Castrillb, L., Gonzalez, L. & Shafritz, D. A. Regulation of hepatic and non-hepatic apolipoprotein gene expression during liver regeneration A-I and E. Biochim. Biophys. Acta 1167, 37–42 (1993).
Mathurin, P. et al. Quantification of apolipoprotein A-I and B messenger RNA in heavy drinkers according to liver disease. Hepatology 23, 44–51 (1996).
Cho, Y. E. et al. Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury. PLoS ONE 12, 1–23 (2017).
Yang, L. et al. Angiopoietin-like protein 4 is a high-density lipoprotein (HDL) component for HDL metabolism and function in nondiabetic participants and type-2 diabetic patients. J. Am. Heart Assoc. 6, 1–15 (2017).
Lichtenstein, L. et al. Angptl4 upregulates cholesterol synthesis in liver via inhibition of LPL- and HL-dependent hepatic cholesterol uptake. Arter. Thromb. Vasc. Biol. 27, 2420–2427 (2007).
Matsuura, F. et al. Adiponectin accelerates reverse cholesterol transport by increasing high density lipoprotein assembly in the liver. Biochem. Biophys. Res. Commun. 358, 1091–1095 (2007).
Singh, A. B. et al. A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression. J. Lipid Res. 55, 1397–1407 (2014).
Li, T. & Chiang J. Y. L. Regulation of bile acid and cholesterol metabolism by PPARs. PPAR Res. 2009, 1–15 (2009).
Al, K. et al. Impact of birth weight and postnatal diet on the gut microbiota of young adult guinea pigs. PeerJ 2017, 1–15 (2017).
Erickson, S. K. et al. Hypercholesterolemia and changes in lipid and bile acid metabolism in male and female cyp7A1-deficient mice. J. Lipid Res. 44, 1001–1009 (2003).
Pullinger, C. R. et al. Human cholesterol 7 α -hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype. J. Clin. Invest. 110, 109–117 (2002).
Gordon, D. A. & Jamil, H. Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly. Biochim. Biophys. Acta 1486, 72–83 (2000).
Li, S. et al. The role of oxidative stress and antioxidants in liver diseases. Int. J. Mol. Sci. 16, 26087–26124 (2015).
Buettner, R. et al. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J. Mol. Endocrinol. 36, 485–501 (2006).
Patleviˇ, P., Vašková, J., Švorc, P. Jr, Vaško, L. & Švorc, P. Reactive oxygen species and antioxidant defense in human gastrointestinal diseases. Integr. Med Res. 5, 250–258 (2016).
Aquilano, K., Baldelli, S. & Ciriolo, M. R. Glutathione: new roles in redox signalling for an old antioxidant. Front. Pharmacol. 5, 1–12 (2014).
Raab, E. L., Vuguin, P. M., Stoffers, D. A. & Simmons, R. A. Neonatal exendin-4 treatment reduces oxidative stress and prevents hepatic insulin resistance in intrauterine growth-retarded rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (2009).
Hu, L. et al. Advances dietary nucleotides supplementation during the suckling period improves the antioxidative ability of neonates with intrauterine growth retardation when using a pig model. RSC Adv. 8, 16152–16160 (2018).
Liu, J. et al. Effect of folic acid supplementation on hepatic antioxidant function and mitochondrial-related gene expression in weanling intrauterine growth retarded piglets. Livest. Sci. 146, 123–132 (2012).
Kreidl, E., Oztürk, D., Metzner, T., Berger, W. & Grusch, M. Activins and follistatins: emerging roles in liver physiology and cancer. World J. Hepatol. 1, 17–27 (2009).
Xu, S., Hou, D., Liu, J. & Ji, L. Communications in free radical research age-associated changes in GSH S-transferase gene/proteins in livers of rats. Redox Rep. 23, 213–218 (2018).
Farmer, M. A. & German, R. Z. Sexual dimorphism in the craniofacial growth of the guinea Pig (Cavia porcellus). J. Morphol. 259, 172–181 (2004).
Oliver, A. & Norbert, S. Diversity of social and mating systems in cavies: a review. J. Mammal. 92, 39–53 (2011).
Phelps, T., Snyder, E., Rodriguez, E., Child, H. & Harvey, P. The influence of biological sex and sex hormones on bile acid synthesis and cholesterol homeostasis. Biol. Sex. Differ. 10, 1–12 (2019).
Movérare-Skrtic, S. et al. Dihydrotestosterone treatment results in obesity and altered lipid metabolism in orchidectomized mice. Obesity 14, 662–672 (2006).
Charni-Natan, M., Aloni-Grinstein, R., Osher, E. & Rotter, V. Liver and steroid hormones-can a touch of p53 make a difference? Front. Endocrinol. (Lausanne) 10, 1–9 (2019).
Hutter, D., Kingdom, J. & Jaeggi, E. Causes and mechanisms of intrauterine hypoxia and its impact on the fetal cardiovascular system: a review. Int. J. Pediatr. 2010, 401323 (2010).
Reuss, M. L. & Rudolph, A. M. Distribution and recirculation of umbilical and systemic venous blood flow in fetal lambs during hypoxia. J. Dev. Physiol. 2, 71–81 (1980).
Ring, J. A., Ghabrial, H., Ching, M. S., Smallwood, R. A. & Morgan, D. J. Fetal hepatic drug elimination. Pharmacol. Ther. 84, 429–445 (1999).
Haugen, G., Kiserud, T., Godfrey, K., Crozier, S. & Hanson, M. Portal and umbilical venous blood supply to the liver in the human fetus near term. Ultrasound Obstet. Gynecol. 24, 599–605 (2004).
Tchirikov, M. & Hecher, K. Ductus venosus shunting in the fetal venous circulation: regulatory mechanisms, diagnostic methods and medical importance. Ultrasound Obstet. Gynecol. 27, 452–461 (2006).
Acknowledgements
The authors thank Brad Matushewski for his assistance with animal surgeries and Brian Sutherland for cholesterol measurements. This work was supported by the Canadian Institutes of Health Research (TRHR: Operating Grant #MOP-209113).
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O.S. and T.R.H.R. designed the experiments, analysed data and drafted the manuscript. O.S., K.E.M., C.V., K.W., A.D., L.Z., and T.R.H.R. performed the experiments. All authors reviewed and edited the manuscript.
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Sarr, O., Mathers, K.E., Vanderboor, C. et al. Sex-specific alterations in hepatic cholesterol metabolism in low birth weight adult guinea pigs. Pediatr Res 91, 1078–1089 (2022). https://doi.org/10.1038/s41390-021-01491-w
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DOI: https://doi.org/10.1038/s41390-021-01491-w