robust-mitochondrial-isolation-from-rodent-cardiac-tissue

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Michigan State University

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Global chemical effects of the microbiome include new bile-acid conjugations.

Undernutrition-induced growth restriction in the early stages of life increases the risk of chronic disease in adulthood. Although metabolic impairments have been observed, few studies have characterized the gut microbiome and gut-liver metabolome profiles of growth-restricted animals during early-to-mid-life development. To induce growth restriction, mouse offspring were either born to gestational undernutrition (GUN) or suckled from postnatal undernutrition (PUN) dams fed a protein-restricted diet (8% protein) or control diet (CON; 20% protein) until weaning at postnatal age of 21 days (PN21). At PN21, all mice were fed the CON diet until adulthood (PN80). Livers were collected at PN21 and PN80, and fecal samples were collected weekly starting at PN21 () until PN80 () for gut microbiome and metabolome analyses. PUN mice exhibited the most alterations in gut microbiome and gut and liver metabolome compared with CON mice. These mice had altered fecal microbial β-diversity ( = 0.001) and exhibited higher proportions of  [linear mixed model (LMM)  = 7.1 × 10),  ( = 1.459 × 10),  ( = 0.0003), and lower  ( = 4.1 × 10)]. PUN liver and fecal metabolome had a reduced total bile acid pool ( < 0.01), as well as lower abundance of riboflavin ( = 0.003), amino acids [i.e., methionine ( = 0.0018), phenylalanine ( = 0.0015), and tyrosine ( = 0.0041)], and higher excreted total peptides (LMM  = 0.0064) compared with CON. Overall, protein restriction during lactation permanently alters the gut microbiome into adulthood. Although the liver bile acids, amino acids, and acyl-carnitines recovered, the fecal peptides and microbiome remained permanently altered into adulthood, indicating that inadequate protein intake in a specific time frame in early life can have an irreversible impact on the microbiome and fecal metabolome. Undernutrition-induced early-life growth restriction not only leads to increased disease risk but also permanently alters the gut microbiome and gut-liver metabolome during specific windows of early-life development.

Longitudinal effects of growth restriction on the murine gut microbiome and metabolome.

The objective of this study was to characterize developmental differences in low birth weight (LBW) and normal birth weight (NBW) piglets with or without pre-weaning nutrient restriction using serum metabolomic profile analysis. At farrowing, 112 piglets were identified as LBW (1.22 ± 0.28 kg) or NBW (1.70 ± 0.27 kg) and were randomly assigned to receive normal nutrition (NN) or restricted nutrition (RN) (6 h/day no suckling) from days 2 to 28 post farrow (n = 8 pigs/group). On day 28, piglets were weaned onto a common diet. Fasted blood samples were obtained on days 28 and 56 (n = 8 pigs/group) and were analyzed using quantitative metabolomics via a combination of direct injection mass spectrometry with a reverse-phase LC-MS/MS custom assay. Data were normalized using logarithmic transformation and auto-scaling. Partial least squares discriminant analysis (PLS-DA) was carried out to further explore the differential metabolites among the groups (metaboanalyst.ca) with an integrated enrichment and pathway topography analysis. On day 28, LBW piglets had lower levels of essential amino acids as well as reduced metabolites associated with fatty acid oxidation, glycolysis, and the tri-carboxylic acid (TCA) cycle compared to the NBW group. The overall reduction of metabolites associated with energy production and regulation suggests that LBW vs. NBW are in an energy-survival state. On day 56, LBW pigs had increased utilization of fatty acids and resultant ketone production, evident by increased carnitines, acetoacetate, β-hydroxybutyrate, and glycerol compared to NBW pigs. In addition, compared to the NBW pigs LBW pigs had a consistent decrease in serum glucose and lactate as well as reduced TCA cycle metabolites: pyruvate, succinate, citrate, and α-ketoglutaric acid similar to day 28. Low reliance on glycolysis and the TCA cycle and higher glycerol production in the LBW pigs may indicate impairments in glucose tolerance at 56 d. In summary, LBW piglets appear to have more metabolic alterations in early life, which is not resolved with adequate nutrition or refeeding and may elucidate physiological and metabolic mechanisms of poor growth and life performance compared to NBW pigs later in life.

Serum metabolomic characterization in pigs in relation to birth weight category and neonatal nutrition.

Growth restriction (GR) reduces ribosome abundance and skeletal muscle mass in mice. A reduction in skeletal muscle mass increases the risk of frailty and is associated with high morbidity and mortality rates. As eccentric type exercise increases muscle mass, this investigation aimed to determine if eccentric loading of skeletal muscle via downhill running (DHR) increased muscle mass in GR mice.

The Effect of Downhill Running on Quadriceps Muscle in Growth-Restricted Mice.

Postnatal growth restriction (PGR) can increase the risk of cardiovascular disease (CVD) potentially due to impairments in oxidative phosphorylation (OxPhos) within cardiomyocyte mitochondria. The purpose of this investigation was to determine if PGR impairs cardiac metabolism, specifically OxPhos. FVB (Friend Virus B-type) mice were fed a normal-protein (NP: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2 weeks before mating. LP dams produce ∼20% less milk, and pups nursed by LP dams experience reduced growth into adulthood as compared to pups nursed by NP dams. At birth (PN1), pups born to dams fed the NP diet were transferred to LP dams (PGR group) or a different NP dam (control group: CON). At weaning (PN21), all mice were fed the NP diet. At PN22 and PN80, mitochondria were isolated for respirometry (oxygen consumption rate,  ) and fluorimetry (reactive oxygen species emission,  ) analysis measured as baseline respiration (LEAK) and with saturating ADP (OxPhos). Western blotting at PN22 and PN80 determined protein abundance of uncoupling protein 3, peroxiredoxin-6, voltage-dependent anion channel and adenine nucleotide translocator 1 to provide further insight into mitochondrial function. ANOVAs with the main effects of diet, sex and age with α-level of 0.05 was set a priori. Overall, PGR (7.8 ± 1.1) had significant (P = 0.01) reductions in respiratory control in complex I when compared to CON (8.9 ± 1.0). In general, our results show that PGR led to higher electron leakage in the form of free radical production and reactive oxygen species emission. No significant diet effects were found in protein abundance. The observed reduced respiratory control and increased ROS emission in PGR mice may increase risk for CVD in mice.

Article	Year
Global chemical effects of the microbiome include new bile-acid conjugations. Nature\| PubMed ID: 32103176	2020
Longitudinal effects of growth restriction on the murine gut microbiome and metabolome. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 35658543	2022
Serum metabolomic characterization in pigs in relation to birth weight category and neonatal nutrition. Journal of animal science\| PubMed ID: 36402552	2023
The Effect of Downhill Running on Quadriceps Muscle in Growth-Restricted Mice. Medicine and science in sports and exercise\| PubMed ID: 37486763	2023
Postnatal growth restriction alters myocardial mitochondrial energetics in mice. Experimental physiology\| PubMed ID: 38180279	2024

Melissa Quinn

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