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

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 disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 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 chemistry14. 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.

Postnatal growth restriction alters myocardial mitochondrial energetics in 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, J O 2 ${J_{{{\mathrm{O}}_{\mathrm{2}}}}}$ ) and fluorimetry (reactive oxygen species emission, J H 2 O 2 ${J_{{{\mathrm{H}}_{\mathrm{2}}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) 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.

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

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 (postweaning week 1) until PN80 (postweaning week 5) 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 ß-diversity (P = 0.001) and exhibited higher proportions of Bifidobacteriales [linear mixed model (LMM) P = 7.1 × 10-6), Clostridiales (P = 1.459 × 10-5), Erysipelotrichales (P = 0.0003), and lower Bacteroidales (P = 4.1 × 10-5)]. PUN liver and fecal metabolome had a reduced total bile acid pool (P < 0.01), as well as lower abundance of riboflavin (P = 0.003), amino acids [i.e., methionine (P = 0.0018), phenylalanine (P = 0.0015), and tyrosine (P = 0.0041)], and higher excreted total peptides (LMM P = 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.NEW & NOTEWORTHY 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.

Postnatal Growth Restriction in Mice Alters Cardiac Protein Composition and Leads to Functional Impairment in Adulthood.

Postnatal growth restriction (PGR) increases the risk for cardiovascular disease (CVD) in adulthood, yet there is minimal mechanistic rationale for the observed pathology. The purpose of this study was to identify proteomic differences in hearts of growth-restricted and unrestricted mice, and propose mechanisms related to impairment in adulthood. Friend leukemia virus B (FVB) mouse dams were fed a control (CON: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2 weeks before mating. LP dams produce 20% less milk, inducing growth restriction. At birth (postnatal; PN1), pups born to dams fed the CON diet were switched to LP dams (PGR group) or a different CON dam. At PN21, a sub-cohort of CON (n = 3 males; n = 3 females) and PGR (n = 3 males; n = 3 females) were euthanized and their proteome analyzed by two-dimensional differential in-gel electrophoresis (2D DIGE) and mass spectroscopy. Western blotting and silver nitrate staining confirmed 2D DIGE results. Littermates (CON: n = 4 males and n = 4 females; PGR: n = 4 males and n = 4 females) were weaned to the CON diet. At PN77, echocardiography measured cardiac function. At PN80, hearts were removed for western blotting to determine if differences persisted into adulthood. 2D DIGE and western blot confirmation indicated PGR had reductions in p57kip2, Titin (Ttn), and Collagen (Col). At PN77, PGR had impaired cardiac function as measured by echocardiography. At PN80, western blots of p57kip2 showed protein abundance recovered from PN21. PN80 silver staining of large molecular weight proteins (Ttn and Col) was reduced in PGR. PGR reduces cell cycle activity at PN21, which is recovered in adulthood. However, collagen fiber networks are altered into adulthood.

The Effect of an Early Life Motor Skill Intervention on Physical Activity in Growth-Restricted Mice.

INTRODUCTION: Early life growth restriction significantly increases the risk of adulthood physical inactivity and thereby chronic disease incidence. Improvements in motor skill acquisition could result in greater physical activity engagement in the growth-restricted population, thus reducing chronic disease risk. The purpose of this study was to implement an early life motor training intervention to improve physical activity engagement in control and growth-restricted mice. METHODS: Mice were growth restricted in early life utilizing a validated nutritive model or remained fully nourished in early life as a control. All mice were tested throughout early life for various components of motor skill acquisition. On postnatal day 10, mice were randomly assigned to engage in an early life motor skill intervention daily until postnatal day 21 or remained as a sedentary control. All mice were given access to an in-cage running wheel from postnatal days 45-70. RESULTS: Growth-restricted group (PGR) mice had impaired trunk and postural control, coordination/vestibular development, and hindlimb strength in early life compared with control mice. There were no differences in wheel running behavior between the trained and sedentary mice, although control mice ran at a faster average speed compared with PGR mice. Control female mice ran more than PGR female mice during the week 2 dark cycle. CONCLUSIONS: Early life growth restriction reduced motor skill attainment throughout early life, which may be associated with reduced ability to engage in physical activity in adulthood. The early life motor skill intervention did not elicit changes in body weight or physical activity engagement in control or PGR mice, indicating that a more intense/different intervention specifically targeting skeletal muscle may be necessary to counteract the detrimental effects of early life growth restriction.

Associations of an HDL apolipoproteomic index with cardiometabolic risk factors before and after exercise training in the HERITAGE Family Study.

BACKGROUND AND AIMS: Previous studies have derived and validated an HDL apolipoproteomic score (pCAD) that predicts coronary artery disease (CAD) risk. However, the associations between pCAD and markers of cardiometabolic health in healthy adults are not known, nor are the effects of regular exercise on pCAD. METHODS: A total of 641 physically inactive adults free of cardiovascular disease from the HERITAGE Family Study completed 20 weeks of exercise training. The pCAD index (range 0-100) was calculated using measurements of apolipoproteins A-I, C-I, C-II, C-III, and C-IV from ApoA-I-tagged serum (higher index = higher CAD risk). The associations between pCAD index and cardiometabolic traits at baseline and their training responses were assessed with Spearman correlation and general linear models. A Bonferroni correction of p < 8.9 × 10-04 was used to determine statistical significance. RESULTS: The mean ± SD baseline pCAD index was 29 ± 32, with 106 (16.5 %) participants classified as high CAD risk. At baseline, pCAD index was positively associated with blood pressure, systemic inflammation, and body composition. HDL size, VO2max, and HDL-C were negatively associated with pCAD index at baseline. Of those classified as high CAD risk at baseline, 52 (49 %) were reclassified as normal risk after training. Following training, pCAD index changes were inversely correlated (p < 1.4 × 10-04) with changes in HDL-C, HDL size, and LDL size. CONCLUSIONS: A higher pCAD index was associated with a worse cardiometabolic profile at baseline but improved with regular exercise. The results from this study highlight the potential role of HDL apolipoproteins as therapeutic targets for lifestyle interventions, particularly in high-risk individuals.

Selectively breeding for high voluntary physical activity in female mice does not bestow inherent characteristics that resemble eccentric remodeling of the heart, but the mini-muscle phenotype does.

Physical activity engagement results in a variety of positive health outcomes, including a reduction in cardiovascular disease risk partially due to eccentric remodeling of the heart. The purpose of this investigation was to determine if four replicate lines of High Runner mice that have been selectively bred for voluntary exercise on wheels have a cardiac phenotype that resembles the outcome of eccentric remodeling. Adult females (average age 55 days) from the 4 High Runner and 4 non-selected control lines were anaesthetized via vaporized isoflurane, then echocardiographic images were collected and analyzed for structural and functional differences. High Runner mice in general had lower ejection fractions compared to control mice lines (2-tailed p â€‹= â€‹0.023 6) and tended to have thicker walls of the anterior portion of the left ventricle (p â€‹= â€‹0.065). However, a subset of the High Runner individuals, termed mini-muscle mice, had greater ejection fraction (p â€‹= â€‹0.000 6), fractional shortening percentage (p â€‹< â€‹0.000 1), and ventricular mass at dissection (p â€‹< â€‹0.002 7 with body mass as a covariate) compared to non-mini muscle mice. Mice from replicate lines bred for high voluntary exercise did not all have inherent positive cardiac functional or structural characteristics, although a genetically unique subset of mini-muscle individuals did have greater functional cardiac characteristics, which in conjunction with their previously described peripheral aerobic enhancements (e.g., increased capillarity) would partially account for their increased V˙ O2max.

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

INTRODUCTION: 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. METHODS: Mice were growth-restricted either gestational undernutrition (GUN, n = 8 litters), postnatal undernutrition (PUN, n = 8 litters), or were not restricted (CON, n = 8 litters) via a validated cross-fostering nutritive model. On postnatal day (PN) 21, all mice were weaned to a healthy diet, isolating the period of GR to early life as seen in humans. At PN45, mice were assigned to either a DHR (CON, n = 4 litters; GUN, n = 4 litters; PUN, n = 4 litters) or sedentary (SED: CON, n = 4 litters; GUN, n = 4 litters; PUN, n = 4 litters) group. Downhill running (16% decline: 18 m·min -1 ) was performed in 30-min bouts, three times per week, for 12 wk on a rodent treadmill. At PN129, the quadriceps femoris was dissected and evaluated for mass, myofiber size and type, and molecular markers of growth. RESULTS: Following training, CON-DHR mice having larger cells than CON-SED, GUN-SED, PUN-SED, and PUN-DHR mice ( P < 0.05). The PUN group (as compared with CON) had reduced body mass ( P < 0.001), upstream binding factor abundance ( P = 0.012), phosphor-mTOR ( P < 0.001), and quadriceps mass ( P = 0.02). The GUN and PUN groups had increased MuRF1 abundance ( P < 0.001) compared with CON ( P < 0.001). CONCLUSIONS: The blunted response to training suggests GR mice may have anabolic resistance when exposed to eccentric type exercise.

C-reactive protein and telomerase reverse transcriptase (TERT) associate with chronic disease markers in a sample from low-income neighborhoods in Detroit, Michigan.

Racial and ethnic minorities in economically deprived inner cities experience high rates of chronic diseases compared to neighborhoods with higher socioeconomic status (SES). However, these economically deprived populations are understudied in terms of biomarkers associated with chronic disease risk which include C-reactive protein (CRP), telomerase reverse transcriptase (TERT), and glycosylated hemoglobin (A1C). We examined relationships between CRP and TERT and chronic disease indicators (body mass index [BMI] and A1C) in two low-income, predominantly African American (AA) neighborhoods in Detroit, Michigan. Sixty-nine adults (43 females, 26 males, mean age 46 years [y], standard deviation [SD] â€‹= â€‹15.9) completed a health survey, anthropometry, and finger stick blood tests. A1C was measured using A1CNow test strips, and CRP and TERT levels were measured using enzyme-linked immunosorbent assay (ELISA) with samples extracted from dried blood spots. We examined CRP (mean â€‹= â€‹4.9, SD â€‹= â€‹3.1), TERT (mean â€‹= â€‹32.5, SD â€‹= â€‹15.1), and A1C (mean â€‹= â€‹5.4, SD â€‹= â€‹1.0) by BMI category. We fitted restricted maximum likelihood regression models to evaluate associations between CRP, TERT, BMI, and A1C, after adjustment for demographics and inclusion of a random effect for the neighborhood. In this predominantly AA sample (91%, 63/69), 68% had levels of CRP (means â€‹= â€‹4.8 â€‹mg/L, SD â€‹= â€‹3.0 for AAs; 6.4 â€‹mg/L, SD â€‹= â€‹3.9 for all others) indicative of chronic inflammation (CRP greater than 3 â€‹mg/L). BMI was significantly associated with CRP (p â€‹= â€‹0.004) and TERT (p â€‹= â€‹0.026). TERT levels indicate that being overweight is associated with markers of chromosome remodeling, suggestive of chronic disease. CRP followed a similar trend with overweight individuals having higher inflammation and risk of chronic disease. Our findings warrant further exploration of additional factors that may influence CRP and TERT. Furthermore, examining populations in a more ethnically and/or economically diverse, yet still high proportion minority, sample will fill a knowledge gap in this understudied field.

The Effect of ACL Reconstruction on Involved and Contralateral Limb Vastus Lateralis Morphology and Histology: A Pilot Study.

Quadriceps muscle weakness is a commonly reported issue post anterior cruciate ligament reconstruction (ACLR), with minimal information related to skeletal muscle morphology following surgery. The purpose is to examine the morphological and functional differences in the vastus lateralis muscle from patient's ACLR and contralateral leg. Three physically active ACLR participants were recruited and secured to a dynamometer to perform maximal voluntary isometric knee extension contractions (MVIC) of the ACLR and contralateral limb. Muscle biopsies of the ACLR and contralateral vastus lateralis were performed, then sectioned, and stained for myosin isoforms to determine fiber type. Confocal images were acquired, and ImageJ software was used to determine the fiber type and cross-sectional area (CSA). There was a significant reduction in CSA of the type IIa and type IIx muscle fiber cells between healthy (IIa: 7,718 ± 1,295 µm2; IIx; 5,800 ± 601 µm2) and ACLR legs (IIa: 4,139 ± 709 µm2; IIx: 3,708 ± 618 µm2) (p < 0.05), while there was no significant difference in knee extension MVIC torque between legs (healthy limb: 2.42 ± 0.52 Nm/kg; ACLR limb: 2.05 ± 0.24 Nm/kg, p = 0.11). The reduction in the cross-sectional area of the ACLR type II fibers could impair function and increase secondary injury risk.

Physical Activity Engagement Worsens Health Outcomes and Limits Exercise Capacity in Growth-restricted Mice.

INTRODUCTION: A total of 161 million children a year are growth restricted, leading to a 47% increased risk of chronic disease in adulthood. Physical activity (PA) reduces the risk of mortality from chronic disease. The purpose of the present investigation was to determine the effect of a PA intervention (wheel running) on cardiac and skeletal muscle capacities in gestational (GUN) and postnatal (PUN) growth-restricted mice as compared with nonrestricted controls (CON). METHODS: A low-protein cross-fostering FVB mouse model was used to induce growth restriction during gestation and the first 21 d of postnatal life. Mouse pups were recovered on a healthy diet until mature and provided wheel access for 3 wk. At completion of the PA intervention, mice underwent maximal exercise testing on a treadmill, echocardiography, and skeletal muscle histology. RESULTS: After the PA intervention, CON mice had a 45% improvement in maximal exercise capacity (P = 0.0390) because of cardiac and skeletal muscle adaptations, but GUN and PUN mice did not. Alarmingly, PUN female mice exposed to wheels had 11.45% lower left ventricular volume (P = 0.0540) and 18% lower left ventricle area (P = 0.0585), with blood flow velocities indicative of cardiac fibrosis (GUN had elevated isovolumetric contraction time P = 0.0374; GUN females and PUN males had longer isovolumetric relaxation time P = 0.0703). PUN male mice had mixed skeletal muscle responses with an oxidative shift in the diaphragm (P = 0.0162) but a glycolytic shift in the extensor digitorum longus (P = 0.0647). PUN female mice had a glycolytic shift in the soleus after wheel running. CONCLUSIONS: Unexpectedly, growth-restricted mice were nonresponders to a PA intervention and displayed negative cardiac outcomes.

Early life undernutrition reduces maximum treadmill running capacity in adulthood in mice.

Undernutrition during early life causes chronic disease with specific impairments to the heart and skeletal muscle. The purpose of this study was to determine the effects of early life undernutrition on adult exercise capacity as a result of cardiac and skeletal muscle function. Pups were undernourished during gestation (GUN) or lactation (PUN) using a cross-fostering nutritive mouse model. At postnatal day 21, all mice were weaned and refed a control diet. At postnatal day 67, mice performed a maximal treadmill test. Echocardiography and Doppler blood flow analysis was performed at postnatal day 72, following which skeletal muscle cross-sectional area (CSA) and fiber type were determined. Maximal running capacity was reduced (diet: P = 0.0002) in GUN and PUN mice. Left ventricular mass (diet: P = 0.03) and posterior wall thickness during systole (diet × sex: P = 0.03) of GUN and PUN mice was reduced, causing PUN mice to have reduced (diet: P = 0.04) stroke volume. Heart rate of GUN mice showed a trend (diet: P = 0.07) towards greater resting values than other groups. PUN mice had greater CSA of soleus fibers. PUN had a reduced (diet: P = 0.03) proportion of type-IIX fibers in the extensor digitorum longus (EDL) and a greater (diet: P = 0.008) percentage of type-IIB fibers in the EDL. In conclusion, gestational and postnatal undernourishment impairs exercise capacity.

The Effect of Growth Restriction on Voluntary Physical Activity Engagement in Mice.

INTRODUCTION: The purpose of this study was to determine the effect of growth restriction on the biological regulation of physical activity. METHODS: Using a cross-fostering, protein-restricted nutritive model, mice were growth-restricted during either gestation (GUN; N = 3 litters) or postnatal life (PUN; N = 3 litters). At 21 d of age, all mice pups were weaned and fed a nonrestrictive healthy diet for the remainder of the study. At 45 d of age, mice were individually housed in cages with free moving running wheels to assess physical activity engagement. At day 70, mice were euthanized, and the nucleus accumbens was analyzed for dopamine receptor 1 expression. Skeletal muscle fiber type and cross-sectional area of the soleus, extensor digitorom longus, and diaphragm were analyzed by immunohistochemistry. The soleus from the other hindleg was evaluated for calsequestrin 1 and annexin A6 expression. RESULTS: The PUN female mice (15,365 ± 8844 revolutions per day) had a reduction (P = 0.0221) in wheel revolutions per day as compared with the GUN (38,667 ± 8648 revolutions per day) and CON females (36,421.0 ± 6700 revolutions per day). The PUN female mice also expressed significantly higher dopamine receptor 1 compared (P = 0.0247) to the other groups. The PUN female soleus had a higher expression of calsequestrin 1, along with more type IIb fibers (P = 0.0398). CONCLUSIONS: Growth restriction during lactation reduced physical activity in female mice by reducing the central drive to be active and displayed a more fatigable skeletal muscle phenotype.