Non-alcoholic fatty liver disease (NAFLD) is an important co-morbidity associated with obesity and a precursor to steatohepatitis. However, the contributions of gestational and early life influences ...on development of NAFLD and NASH remain poorly appreciated.
Two independent studies were performed to examine whether maternal over-nutrition via exposure to high fat diet (HFD) leads to exacerbated hepatic responses to post-natal HFD and methionine choline deficient (MCD) diets in the offspring. Offspring of both control diet- and HFD-fed dams were weaned onto control and HFD, creating four groups.
When compared to their control diet-fed littermates, offspring of HF-dams weaned onto HFD gained greater body weight; had increased relative liver weight and showed hepatic steatosis and inflammation. Similarly, this group revealed significantly greater immune response and pro-fibrogenic gene expression via RNA-seq. In parallel, 7-8 week old offspring were challenged with either control or MCD diets for 3 weeks. Responses to MCD diets were also exacerbated due to maternal HFD as seen by gene expression of classical pro-fibrogenic genes. Quantitative genome-scale DNA methylation analysis of over 1 million CpGs showed persistent epigenetic changes in key genes in tissue development and metabolism (Fgf21, Ppargc1β) with maternal HFD and in cell adhesion and communication (VWF, Ephb2) in the combination of maternal HFD and offspring MCD diets. Maternal HFD also influenced gut microbiome profiles in offspring leading to a decrease in α-diversity. Linear regression analysis revealed association between serum ALT levels and Coprococcus, Coriobacteriacae, Helicobacterioceae and Allobaculum.
Our findings indicate that maternal HFD detrimentally alters epigenetic and gut microbiome pathways to favor development of fatty liver disease and its progressive sequelae.
The contributions of maternal diet and obesity in shaping offspring microbiome remain unclear. Here we employed a mouse model of maternal diet-induced obesity via high-fat diet feeding (HFD, 45% fat ...calories) for 12 wk prior to conception on offspring gut microbial ecology. Male and female offspring were provided access to control or HFD from weaning until 17 wk of age. Maternal HFD-associated programming was sexually dimorphic, with male offspring from HFD dams showing hyper-responsive weight gain to postnatal HFD. Likewise, microbiome analysis of offspring cecal contents showed differences in α-diversity, β-diversity and higher Firmicutes in male compared to female mice. Weight gain in offspring was significantly associated with abundance of Lachnospiraceae and Clostridiaceae families and Adlercreutzia, Coprococcus and Lactococcus genera. Sex differences in metagenomic pathways relating to lipid metabolism, bile acid biosynthesis and immune response were also observed. HFD-fed male offspring from HFD dams also showed worse hepatic pathology, increased pro-inflammatory cytokines, altered expression of bile acid regulators (Cyp7a1, Cyp8b1 and Cyp39a1) and serum bile acid concentrations. These findings suggest that maternal HFD alters gut microbiota composition and weight gain of offspring in a sexually dimorphic manner, coincident with fatty liver and a pro-inflammatory state in male offspring.
Neonatal diet has a large influence on child health and might modulate changes in fecal microbiota and metabolites.
The aim is to investigate fecal microbiota and metabolites at different ages in ...infants who were breastfed (BF), received dairy-based milk formula (MF), or received soy-based formula (SF).
Fecal samples were collected at 3 (n = 16, 12, and 14, respectively), 6 (n = 20, 19, and 15, respectively), 9 (n = 12, 11, and 12, respectively), and 12 mo (n = 14, 14, and 15, respectively) for BF, MF, and SF infants. Infants that breastfed until 9 mo and switched to formula were considered as no longer breastfeeding at 12 mo. Microbiota data were obtained using 16S ribosomal RNA sequencing. Untargeted metabolomics was conducted using a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. The data were analyzed using R (version 3.6.0) within the RStudio (version 1.1.463) platform.
At 3, 6, and 9 mo of age BF infants had the lowest α-diversity, SF infants had the highest diversity, and MF was intermediate. Bifidobacterium was 2.6- to 5-fold lower in SF relative to BF infants through 1 y of life. An unidentified genus from Ruminococcaceae higher in the SF (2%) than in the MF (0.4%) and BF (0.08%) infants at 3 mo of age was observed. In BF infants higher levels of butyric acid, d-sphingosine, kynurenic acid, indole-3-lactic acid, indole-3-acetic acid, and betaine were observed than in MF and SF infants. At 3 mo Ruminococcaceae was positively correlated to azelaic, gentisic, isocitric, sebacic, and syringic acids. At 6 mo Oscillospira was negatively correlated with 3-hydroxybutyric-acid, hydroxy-hydrocinnamic acid, and betaine whereas Bifidobacterium was negatively associated with 5-hydroxytryptamine. At 12 mo of age, Lachnospiraceae was negatively associated with hydroxyphenyllactic acid.
Infant diet has a large impact on the fecal microbiome and metabolome in the first year of life. This study was registered at clinicaltrials.gov as NCT00616395.
Myoglobin (Mb), besides its roles as an oxygen (O2) carrier/storage protein and nitric oxide NO scavenger/producer, may participate in lipid trafficking and metabolite binding. Our recent findings ...have shown that O2 is released from oxy-Mb upon interaction with lactate (LAC, anerobic glycolysis end-product). Since pyruvate (PYR) is structurally similar and metabolically related to LAC, we investigated the effects of PYR (aerobic glycolysis end-product) on Mb using isothermal titration calorimetry, circular dichroism, and O2-kinetic studies to evaluate PYR affinity toward Mb and to compare the effects of PYR and LAC on O2 release kinetics of oxy-Mb. Similar to LAC, PYR interacts with both oxy- and deoxy-Mb with a 1:1 stoichiometry. Time-resolved circular dichroism spectra revealed that there are no major conformational changes in the secondary structures of oxy- or deoxy-Mb during interactions with PYR or LAC. However, we found contrasting results with respect to binding affinities and substrate preference, where PYR has higher affinity toward deoxy-Mb when compared with LAC (which prefers oxy-Mb). Furthermore, PYR interaction with oxy-Mb releases a significantly lower amount of O2 than LAC. Taken together, our findings support the hypothesis that glycolytic end-products play a distinctive role in the Mb-rich tissues by serving as novel regulators of O2 availability, and/or by impacting other activities related to oxy-/deoxy-Mb toggling in resting vs. exercised or metabolically activated conditions.
Myoglobin, besides its role in oxygen turnover, has gained recognition as a potential regulator of lipid metabolism. Previously, we confirmed the interaction of fatty acids and acylcarnitines with ...Oxy-Myoglobin, using both molecular dynamic simulations and Isothermal Titration Calorimetry studies. However, those studies were limited to testing only the binding sites derived from homology to fatty acid binding proteins and predictions using automated docking. To explore the entry mechanisms of the lipid ligands into myoglobin, we conducted molecular dynamic simulations of murine Oxy- and Deoxy-Mb structures with palmitate or palmitoylcarnitine starting at different positions near the protein surface. The simulations indicated that both ligands readily (under ∼10–20 ns) enter the Oxy-Mb structure through a dynamic area (“portal region”) near heme, known to be the entry point for small molecule gaseous ligands like O2, CO and NO. The entry is not observed with Deoxy-Mb where lipid ligands move away from protein surface, due to a compaction of the entry portal and the heme-containing crevice in the Mb protein upon O2 removal. The results suggest quick spontaneous binding of lipids to Mb driven by hydrophobic interactions, strongly enhanced by oxygenation, and consistent with the emergent role of Mb in lipid metabolism.
Myoglobin (Mb)-mediated oxygen (O
) delivery and dissolved O
in the cytosol are two major sources that support oxidative phosphorylation. During intense exercise, lactate (LAC) production is elevated ...in skeletal muscles as a consequence of insufficient intracellular O
supply. The latter results in diminished mitochondrial oxidative metabolism and an increased reliance on nonoxidative pathways to generate ATP. Whether or not metabolites from these pathways impact Mb-O
associations remains to be established. In the present study, we employed isothermal titration calorimetry, O
kinetic studies, and UV-Vis spectroscopy to evaluate the LAC affinity toward Mb (oxy- and deoxy-Mb) and the effect of LAC on O
release from oxy-Mb in varying pH conditions (pH 6.0-7.0). Our results show that LAC avidly binds to both oxy- and deoxy-Mb (only at acidic pH for the latter). Similarly, in the presence of LAC, increased release of O
from oxy-Mb was detected. This suggests that with LAC binding to Mb, the structural conformation of the protein (near the heme center) might be altered, which concomitantly triggers the release of O
. Taken together, these novel findings support a mechanism where LAC acts as a regulator of O
management in Mb-rich tissues and/or influences the putative signaling roles for oxy- and deoxy-Mb, especially under conditions of LAC accumulation and lactic acidosis.
Early infant diet influences postnatal gut microbial development, which in turn can modulate the developing immune system.
The aim of this study was to characterize diet-specific bioregional ...microbiota differences in piglets fed either human breast milk (HM) or infant formula.
Male piglets (White Dutch Landrace Duroc) were raised on HM or cow milk formula (MF) from postnatal day (PND) 2 to PND 21 and weaned to an ad libitum diet until PND 51. Piglets were euthanized on either PND 21 or PND 51, and the gastrointestinal contents were collected for 16sRNA sequencing. Data were analyzed using the Quantitative Insight into Microbial Ecology. Diversity measurements (Chao1 and Shannon) and the Wald test were used to determine relative abundance.
At PND 21, the ileal luminal region of HM-fed piglets showed lower Chao1 operational taxonomic unit diversity, while Shannon diversity was lower in cecal, proximal colon (PC), and distal colon (DC) luminal regions, relative to MF-fed piglets. In addition, at PND 51, the HM-fed piglets had lower genera diversity within the jejunum, ileum, PC, and DC luminal regions, relative to MF-fed piglets. At PND 21, Turicibacter was 4- to 5-fold lower in the HM-fed piglets’ ileal, cecal, PC, and DC luminal regions, relative to the MF-fed piglets. Campylobacter is 3- to 6-fold higher in HM-fed piglets duodenal, ileal, cecal, PC, and DC luminal regions, in comparison to MF-fed piglets. Furthermore, the large intestine (cecum, PC, and rectum) luminal region of HM-fed piglets showed 4- to 7-fold higher genera that belong to class Bacteroidia, in comparison to MF-fed piglets at PND 21. In addition, at PND 51 distal colon lumen of HM-fed piglets showed 1.5-fold higher genera from class Bacteroidia than the MF-fed piglets.
In the large intestinal regions (cecum, PC, and rectum), MF diet alters microbiota composition, relative to HM diet, with sustained effects after weaning from the neonatal diet. These microbiota changes could impact immune system and health outcomes later in life.
The gut microbiota plays a critical role in energy homeostasis and its dysbiosis is associated with obesity. Maternal high-fat diet (HFD) and β-adrenergic stimuli alter the gut microbiota ...independently; however, their collective regulation is not clear. To investigate the combined effect of these factors on offspring microbiota, 20-week-old offspring from control diet (17% fat)- or HFD (45% fat)-fed dams received an injection of either vehicle or β3-adrenergic agonist CL316,243 (CL) for 7 days and then cecal contents were collected for bacterial community profiling. In a follow-up study, a separate group of mice were exposed to either 8 °C or 30 °C temperature for 7 days and blood serum and cecal contents were used for metabolome profiling. Both maternal diet and CL modulated the gut bacterial community structure and predicted functional profiles. Particularly, maternal HFD and CL increased the Firmicutes/Bacteroidetes ratio. In mice exposed to different temperatures, the metabolome profiles clustered by treatment in both the cecum and serum. Identified metabolites were enriched in sphingolipid and amino acid metabolism in the cecum and in lipid and energy metabolism in the serum. In summary, maternal HFD altered offspring's response to CL and altered microbial composition and function. An independent experiment supported the effect of thermogenic challenge on the bacterial function through metabolome change.
The gut microbiome plays a critical role in the onset and progression of obesity and the metabolic syndrome. However, it is not well documented whether the cecal vs. the fecal microbiome is more ...relevant when assessing their contributions to these diseases. Here, we amplified the V4 region of the 16S rRNA gene from cecal and fecal samples of female Ossabaw swine fed a low-fat control diet (10.5% fat, n = 4) or Western diet (43.0% fat, 17.8% high fructose corn syrup, 2% cholesterol; n = 3) for 36 wk. Obesity significantly lowered alpha-diversity ( P < 0.05), and there was clear separation in beta-diversity between lean and obese pigs, as well as between cecal and fecal samples ( P < 0.05). Obesity dramatically increased ( P < 0.05) the Firmicutes:Bacteroidetes ratio in fecal samples, and Actinobacteria was higher ( P < 0.05) in fecal vs. cecal samples in obese pigs. Cyanobacteria, Proteobacteria, and Fusobacteria were increased ( P < 0.05), while Spirochaetes, Tenericutes, and Verrucomicrobia were decreased ( P < 0.05) in obese vs. lean pigs. Prevotellaceae was reduced ( P < 0.05) in obese fecal vs. cecal samples. Moreover, cecal samples in obese had greater ( P < 0.05) predicted metabolic capacity for glycan biosynthesis and metabolism and LPS biosynthesis compared with fecal. Obese pigs also had greater ( P < 0.05) capacity for carbohydrate metabolism, which was driven by obese fecal rather than cecal samples and was opposite in lean pigs ( P < 0.05). The observed differences in pro-inflammatory microbiota and their metabolic capacity in cecal vs. fecal samples of obese pigs provide new insight into evaluating the microbiome in the pathogenesis of obesity and metabolic disease.
Maternal obesity predisposes offspring to the development of fatty liver and nonalcoholic steatohepatitis. Using a well-established mouse model (C57BL6/J) of maternal high-fat, high sucrose diet ...(HFD) exposure, we characterized epigenomic alterations in adult offspring liver. ChiP-seq for six histone modifications were performed on offspring livers from dams fed either a AIN93G (CON, 7% fat calories, n=11) or HFD (45% fat calories, n=9) for 12 wk prior to pregnancy and through weaning. Offspring of HFD dams, showed a robust gain in repressive H3K27me3 modification mediated by the histone methyltransferase, Ezh2. Since Ezh2 has been implicated in modulating inflammatory signaling we examined if Ezh2 in macrophages contributes to hepatic inflammation following a high-fat palmitate/cholesterol (FPC) diet. We generated myeloid cell-specific Ezh2-knockouts (Ezh2-CKO) by crossing Ezh2-floxed (Ezh2f/f) mice to LysM-Cre expressing mice. At 5 weeks of age Ezh2-CKO and flox-control litter mates were provided ad libitum CON (n=22) or FPC diet (52% fat calories, n=22) for 14 days. At 7 weeks of age, body and liver weights were collected and liver tissue was flash frozen for RNA-seq analyses. Two-way ANOVA of liver to body weight ratios showed a significant diet effect (p<0.0001) , with increased ratios in the FPC group without effects of genotype. Transcriptomic analyses showed that the effects of FPC diet were diminished in CKO mice relative to flox controls (223 vs.132 DEGs in flox and CKO, respectively) with 2DEGs shared between genotypes. Ingenuity Pathway Analysis revealed signaling pathways and processes related to inflammation, macrophage and leukocyte activation, reactive oxygen species, and PPARα that were increased in the flox with FPC diets compared to CKO animals. Collectively, these findings reveal a role for Ezh2 in developmental programing of liver disease and hepatic inflammation, uncovering a novel epigenetic process influencing hepatic disease risk in offspring.
Disclosure
M.L.Ruebel: None. P.Jambal: None. S.V.Chintapalli: None. J.Chen: None. K.Shankar: None.
Funding
U.S. Department of Agriculture-ARS-CRIS (6251-51000-005-00D) NIH (P30DK048520-27, R01HD102726-01A1) Funds from the Department of Pediatrics, University of ColoradoAnschutz Medical Campus and the Anschutz Health and Wellness Center.
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