mARC1 Is the Main Contributor to Metabolic Reduction of N-Hydroxyurea.

N-Hydroxyurea has been known since the 1960s as an antiproliferative drug and is used both in oncology and for treatment of hematological disorders such as sickle cell anemia where very high daily doses are administered. It is assumed that the cellular effect of N-hydroxyurea is caused by inhibition of ribonucleotide reductase, while alternative mechanisms, e.g., generation of nitric oxide, have also been proposed. Despite its many therapeutic applications, the metabolism of hydroxyurea is largely unexplored. The major elimination pathway of N-hydroxyurea is the reduction to urea. Since the mitochondrial amidoxime reducing component (mARC) is known for its N-reductive activity, we investigated the reduction of NHU by this enzyme system. This study presents in vitro and in vivo evidence that this reductive biotransformation is specifically mediated by the mARC1. Inactivation by mARC1 is a possible explanation for the high doses of NHU required for treatment.

mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.

Soluble Guanylyl Cyclase Activator BI 685509 Reduces Portal Hypertension and Portosystemic Shunting in a Rat Thioacetamide-Induced Cirrhosis Model.

Portal hypertension (PT) commonly occurs in cirrhosis. Nitric oxide (NO) imbalance contributes to PT via reduced soluble guanylyl cyclase (sGC) activation and cGMP production, resulting in vasoconstriction, endothelial cell dysfunction, and fibrosis. We assessed the effects of BI 685509, an NO-independent sGC activator, on fibrosis and extrahepatic complications in a thioacetamide (TAA)-induced cirrhosis and PT model. Male Sprague-Dawley rats received TAA twice-weekly for 15 weeks (300-150 mg/kg i.p.). BI 685509 was administered daily for the last 12 weeks (0.3, 1, and 3 mg/kg p.o.; n = 8-11 per group) or the final week only (Acute, 3 mg/kg p.o.; n = 6). Rats were anesthetized to measure portal venous pressure. Pharmacokinetics and hepatic cGMP (target engagement) were measured by mass spectrometry. Hepatic Sirius Red morphometry (SRM) and alpha-smooth muscle actin (αSMA) were measured by immunohistochemistry; portosystemic shunting was measured using colored microspheres. BI 685509 dose-dependently increased hepatic cGMP at 1 and 3 mg/kg (3.92 ± 0.34 and 5.14 ± 0.44 versus 2.50 ± 0.19 nM in TAA alone; P < 0.05). TAA increased hepatic SRM, αSMA, PT, and portosystemic shunting. Compared with TAA, 3 mg/kg BI 685509 reduced SRM by 38%, αSMA area by 55%, portal venous pressure by 26%, and portosystemic shunting by 10% (P < 0.05). Acute BI 685509 reduced SRM and PT by 45% and 21%, respectively (P < 0.05). BI 685509 improved hepatic and extrahepatic cirrhosis pathophysiology in TAA-induced cirrhosis. These data support the clinical investigation of BI 685509 for PT in patients with cirrhosis. SIGNIFICANCE STATEMENT: BI 685509 is an NO-independent sGC activator that was tested in a preclinical rat model of TAA-induced nodular, liver fibrosis, portal hypertension, and portal systemic shunting. BI 685509 reduced liver fibrosis, portal hypertension, and portal-systemic shunting in a dose-dependent manner, supporting its clinical assessment to treat portal hypertension in patients with cirrhosis.

Poor maternal diet during gestation alters offspring muscle proteome in sheep.

Poor maternal nutrition during gestation can result in reduced offspring muscle growth and altered muscle metabolism. We hypothesized that over- or restricted-nutrition during gestation would alter the longissimus dorsi muscle (LM) proteome of offspring. Pregnant ewes were fed 60% (restricted), 100% (control), or 140% (over) of National Research Council requirements for total digestible nutrients from day 30 of gestation until parturition. Fetal (RES, CON, OVER) LM were collected at days 90 and 135 of gestation, or from offspring within 24 h of birth. Sarcoplasmic proteins were isolated, trypsin digested, and subjected to multiplexed, label-based quantitative mass spectrometry analysis integrating tandem mass tag technology. Differential expression of proteins was identified by ANOVA followed by Tukey's HSD post hoc tests, and regularized regression via the elastic net. Significance was set at P < 0.05. Over-represented pathways containing differentially expressed proteins were identified by Reactome and included metabolism of proteins, immune system, cellular response to stress/external stimuli, developmental biology, and infectious disease. As a result of maternal diet, a total of 312 proteins were differentially expressed (day 90 = 89 proteins; day 135 = 115 proteins; birth = 131 proteins). Expression of eukaryotic initiation factor (EIF) 2S3, EIF3L, and EIF4G2 was lower in OVER fetuses at day 90 of gestation (P < 0.05). Calcineurin A and mitogen-activated protein kinase 1 were greater in RES fetuses at day 90 (P < 0.04). At day 135 of gestation, pyruvate kinase and lactate dehydrogenase A expression were greater in OVER fetuses than CON (P < 0.04). Thioredoxin expression was greater in RES fetuses relative to CON at day 135 (P = 0.05). At birth, proteins of the COP9 signalosome complex were greater in RES offspring relative to OVER (P < 0.05). Together, these data indicate that protein degradation and synthesis, metabolism, and oxidative stress are altered in a time and diet-specific manner, which may contribute to the phenotypic and metabolic changes observed during fetal development and postnatal growth.

Poor maternal diet during gestation results in changes in body composition and metabolism in the offspring. Here, we demonstrate that over- and restricted-feeding during gestation alter global protein expression in the longissimus dorsi muscle of offspring during gestation and just after birth. These protein changes are related to protein synthesis and degradation, stress responses, metabolism, and oxidative stress. Proteins related to the initiation of protein translation were increased in offspring of over-fed dams at mid-gestation, while changes in abundance of enzymes associated with metabolism were altered in late gestation and just after birth. In offspring of restricted-fed ewes, proteins relating to cell signaling were increased at mid-gestation, while again, changes in late gestation and birth were related to metabolism, protein degradation, and stress responses. Together, these may provide a mechanism by which poor maternal diet during gestation alters the poor growth and development that occurs in these offspring.

Maternal nutrient restriction and over-feeding during gestation alter expression of key factors involved in placental development and vascularization.

Poor maternal nutrition can negatively affect fetal and placental growth and development. However, the mechanism(s) that contribute to altered placenta growth and function are not well understood. We hypothesized that poor maternal diet would impact signaling through the C-X-C motif chemokine ligand (CXCL) 12-CXCL4 axis and/or placental expression of the insulin-like growth factor (IGF) axis. Using our established sheep model of poor maternal nutrition, we examined the effects of restricted- and over-feeding on ewe placentome gene and protein expression. Specifically, ewes were fed a control (CON; 100%), restricted (RES; 60%), or over (OVER; 140%) diet beginning at day 30.2 ± 0.02 of gestation, and samples were collected at days 45, 90, and 135 of gestation, representing periods of active placentation, peak placental growth, and near term, respectively. Placentomes were separated into cotyledon and caruncle, and samples snap frozen. Protein was determined by western blot and mRNA expression by real-time PCR. Data were analyzed by ANOVA and significance determined at P ≤ 0.05. Ewes fed a RES diet had decreased CXCL12 and vascular endothelial growth factor (VEGF), and increased tumor necrosis factor (TNF)α protein compared with CON ewes in caruncle at day 45 (P ≤0.05). In day 45 cotyledon, CXCR7 protein was increased and mTOR was decreased in RES relative to CON (P ≤0.05). At day 90, CXCR4 and CXCR7 were reduced in RES caruncle compared with CON, whereas VEGF was reduced and mTOR increased in cotyledon of RES ewes relative to CON (P ≤0.05). In OVER caruncle, at day 45 CXCR4 and VEGF were reduced and at day 90 CXCR4, CXCR7, and TNFα were reduced in caruncle compared with CON (P ≤0.05). There was no observed effect of OVER diet on protein abundance in the cotyledon (P > 0.05). Expression of IGF-II mRNA was increased in OVER at day 45 and IGFBP-3 was reduced in RES at day 90 in caruncle relative to CON (P ≤0.05). Maternal diet did not alter placentome diameter or weight (P > 0.05). These findings suggest that restricted- and over-feeding negatively impact protein and mRNA expression of key chemokines and growth factors implicated in proper placenta development and function.

Too little or too much food during gestation can lead to poor growth and health of the resulting offspring. The placenta is an important source of nutrient supply for the fetus and poor maternal diet can impair placenta growth and function. Although placental development and function are well studied, the mechanisms by which maternal diet can affect placental growth and fetal development are not well understood. Based on our previous findings that specific proteins are important regulators of placental growth and function, we used a sheep model of poor maternal nutrition to demonstrate that protein abundance of these factors is altered in the placenta. These findings demonstrate potential mechanism by which maternal diet can affect the placenta and thereby impact fetal growth.

Tissue-specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep.

Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and LDH protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, ß-cell area, or genes regulating ß-cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia-induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization.NEW & NOTEWORTHY Hypoxemia lowered fetal glucose oxidation rates, based on severity of hypoxemia, and increased lactate production. This was supported by tissue-specific metabolic responses that may result from increased norepinephrine and cortisol concentrations, which decrease pancreatic insulin secretion and insulin concentrations and decrease glucose utilization. This highlights the vulnerability of metabolic pathways in the fetus and demonstrates that constrained glucose oxidation may represent an early event in response to sustained hypoxemia and fetal growth restriction.

Fetal Sex Does Not Impact Placental Blood Flow or Placental Amino Acid Transfer in Late Gestation Pregnant Sheep With or Without Placental Insufficiency.

Pregnant sheep have been used to model complications of human pregnancies including placental insufficiency and intrauterine growth restriction. Some of the hallmarks of placental insufficiency are slower uterine and umbilical blood flow rates, impaired placental transport of oxygen and amino acids, and lower fetal arterial concentrations of anabolic growth factors. An impact of fetal sex on these outcomes has not been identified in either human or sheep pregnancies. This is likely because most studies measuring these outcomes have used small numbers of subjects or animals. We undertook a secondary analysis of previously published data generated by our laboratory in late-gestation (gestational age of 133 ± 0 days gestational age) control sheep (n = 29 male fetuses; n = 26 female fetuses; n = 3 sex not recorded) and sheep exposed to elevated ambient temperatures to cause experimental placental insufficiency (n = 23 male fetuses; n = 17 female fetuses; n = 1 sex not recorded). The primary goal was to determine how fetal sex modifies the effect of the experimental insult on outcomes related to placental blood flow, amino acid and oxygen transport, and fetal hormones. Of the 112 outcomes measured, we only found an interaction between fetal sex and experimental insult for the uterine uptake rates of isoleucine, phenylalanine, and arginine. Additionally, most outcomes measured did not show a difference based on fetal sex when adjusting for the impact of placental insufficiency. Exceptions included fetal norepinephrine and cortisol concentrations, which were higher in female compared to male fetuses. For the parameters measured in the current analysis, the impact of fetal sex was not widespread.

Uteroplacental nutrient flux and evidence for metabolic reprogramming during sustained hypoxemia.

Gestational hypoxemia is often associated with reduced birth weight, yet how hypoxemia controls uteroplacental nutrient metabolism and supply to the fetus is unclear. This study tested the effects of maternal hypoxemia (HOX) between 0.8 and 0.9 gestation on uteroplacental nutrient metabolism and flux to the fetus in pregnant sheep. Despite hypoxemia, uteroplacental and fetal oxygen utilization and net glucose and lactate uptake rates were similar in HOX (n = 11) compared to CON (n = 7) groups. HOX fetuses had increased lactate and pyruvate concentrations and increased net pyruvate output to the utero-placenta. In the HOX group, uteroplacental flux of alanine to the fetus was decreased, as was glutamate flux from the fetus. HOX fetuses had increased alanine and decreased aspartate, serine, and glutamate concentrations. In HOX placental tissue, we identified hypoxic responses that should increase mitochondrial efficiency (decreased SDHB, increased COX4I2) and increase lactate production from pyruvate (increased LDHA protein and LDH activity, decreased LDHB and MPC2), both resembling metabolic reprogramming, but with evidence for decreased (PFK1, PKM2), rather than increased, glycolysis and AMPK phosphorylation. This supports a fetal-uteroplacental shuttle during sustained hypoxemia whereby uteroplacental tissues produce lactate as fuel for the fetus using pyruvate released from the fetus, rather than pyruvate produced from glucose in the placenta, given the absence of increased uteroplacental glucose uptake and glycolytic gene activation. Together, these results provide new mechanisms for how hypoxemia, independent of AMPK activation, regulates uteroplacental metabolism and nutrient allocation to the fetus, which allow the fetus to defend its oxidative metabolism and growth.

Maternal Overnutrition During Gestation in Sheep Alters Autophagy Associated Pathways in Offspring Heart.

Poor maternal nutrition during gestation can negatively affect offspring growth, development, and health pre- and post-natally. Overfeeding during gestation or maternal obesity (MO) results in altered metabolism and imbalanced endocrine hormones in animals and humans which will have long-lasting and detrimental effects on offspring growth and health. In this study, we examined the effects of overnutrition during gestation on autophagy associated pathways in offspring heart muscles at two gestational and one early postnatal time point (n = 5 for treated and untreated male and female heart respectively at each time point). Two-way ANOVA was used to analyze the interaction between treatment and sex at each time point. Our results revealed significant interactions of maternal diet by developmental stages for offspring autophagy signaling. Overfeeding did not affect the autophagy signaling at mid-gestation day 90 (GD90) in both male and female offspring while the inflammatory cytokines were increased in GD90 MO male offsrping; however, overfeeding during gestation significantly increased autophagy signaling, but not inflammation level at a later developmental stage (GD135 and day 1 after birth) in both males and females. We also identified a sexual dimorphic response in which female progeny were more profoundly influenced by maternal diet than male progeny regardless of developmental stages. We also determined the cortisol concentrations in male and female hearts at three developmental stages. We did not observe cortisol changes between males and females or between overfeeding and control groups. Our exploratory studies imply that MO alters autophagy associated pathways in both male and female at later developmental stages with more profound effects in female. This finding need be confirmed with larger sample numbers in the future. Our results suggest that targeting on autophagy pathway could be a strategy for correction of adverse effects in offspring of over-fed ewes.

Revitalizing the common peroneal function index for assessing functional recovery following nerve injury.

BACKGROUND AND AIMS: Peripheral nerve injury is common with poor functional recovery and consequent high personal and societal costs. Sciatic nerve transection and assessment of recovery using sciatic functional index (SFI) are widely used. SFI is biologically limited as axonal misdirection of axons supplying flexors and extensors in the hindlimb, after nerve injury can lead to synkinetic innervation and function which does not correspond to the degree of axonal regeneration. METHODS: We reevaluated the use of traditional metrics such as print length (PL), toe spread (TS), and intermediate toe spread (ITS) as well as hock angle at mid-swing as approaches for determining recovery. We used two alternative approaches in discrete cohorts of rats following common peroneal crush injury, transection with repair and critical gap, using transection with ligation as a negative control. We compared walking track analysis (print) with digital capture and kinematics. RESULTS: PL, TS, and ITS varied as expected after injury. The traditional functional index for common peroneal injury using inked prints failed to describe recovery and we derived new indices to describe recovery (all R2  > 0.88, p < .0001) although pre-injury PFI was never attained by any of the models. Kinematic analysis identified hock angle at mid-swing as a useful predictor of recovery (p < .0001). INTERPRETATION: Using complementary approaches.

Effects of chronic hyperinsulinemia on metabolic pathways and insulin signaling in the fetal liver.

The effect of chronic of hyperinsulinemia in the fetal liver is poorly understood. Here, we produced hyperinsulinemia with euglycemia for ∼8 days in fetal sheep [hyperinsulinemic (INS)] at 0.9 gestation. INS fetuses had increased insulin and decreased oxygen and amino acid (AA) concentrations compared with saline-infused fetuses [control (CON)]. Glucose (whole body) utilization rates were increased, as expected, in INS fetuses. In the liver, however, there were few differences in genes and metabolites related to glucose and lipid metabolism and no activation of insulin signaling proteins (Akt and mTOR). There was increased p-AMPK activation and decreased mitochondrial mass (PGC1A expression, mitochondrial DNA content) in INS livers. Using an unbiased multivariate analysis with 162 metabolites, we identified effects on AA and one-carbon metabolism in the INS liver. Expression of the transaminase BCAT2 and glutaminase genes GLS1 and GLS2 was decreased, supporting decreased AA utilization. We further evaluated the roles of hyperinsulinemia and hypoxemia, both present in INS fetuses, on outcomes in the liver. Expression of PGC1A correlated only with hyperinsulinemia, p-AMPK correlated only with hypoxemia, and other genes and metabolites correlated with both hyperinsulinemia and hypoxemia. In fetal hepatocytes, acute treatment with insulin activated p-Akt and decreased PGC1A, whereas hypoxia activated p-AMPK. Overall, chronic hyperinsulinemia produced greater effects on amino acid metabolism compared with glucose and lipid metabolism and a novel effect on one-carbon metabolism in the fetal liver. These hepatic metabolic responses may result from the downregulation of insulin signaling and antagonistic effects of hypoxemia-induced AMPK activation that develop with chronic hyperinsulinemia.

The Effect of Season on Muscle Growth, Fat Deposition, Travel Patterns, and Hoof Growth of Domestic Young Horses.

Our objective was to determine the influence of season (winter, spring, summer, and fall) on travel patterns, hoof growth, and longissimus dorsi muscle (LM) height and fat thickness between 13th and 14th ribs in 16 horses aged <4 years (eight males and eight females) of Morgan, Quarter Horse, and Moriesian breeds. Real-time ultrasound images of LM height and fat thickness as well as measures of hoof growth were obtained at the end of each season. Global positioning system tracking was conducted for four randomly selected days and one storm day in each season. Data were analyzed using a linear mixed model procedure in SAS. Season influenced fat deposition (P < .01) with the greatest increase in fall (P < .05) but had minimal effect on muscle growth. Hoof growth was greatest in summer and least in winter (P < .01). The average distance traveled was greater in spring and summer than in fall and winter (P < .01). The horses moved for less time and traveled less distance on storm days (P < .05) compared with nonstorm days. Young horses also traveled less on storm days, which indicates that it may be especially important to provide shelter for them. It was concluded that season influenced fat deposition, distance traveled, and hoof growth of domestic young horses. A better understanding of these factors could help equine professionals manage young horses more efficiently to benefit the horses' physical well-being.

Poor maternal nutrition during gestation in sheep alters prenatal muscle growth and development in offspring.

Poor maternal nutrition during gestation can have immediate and life-long negative effects on offspring growth and health. In livestock, this leads to reduced product quality and increased costs of production. Based on previous evidence that both restricted- and overfeeding during gestation decrease offspring muscle growth and alter metabolism postnatally, we hypothesized that poor maternal nutrition during gestation would reduce the growth and development of offspring muscle prenatally, reduce the number of myogenic progenitor cells, and result in changes in the global expression of genes involved in prenatal muscle development and function. Ewes were fed a control (100% NRC)-, restricted (60% NRC)-, or overfed (140% NRC) diet beginning on day 30 of gestation until days 45, 90, and 135 of gestation or until parturition. At each time point fetuses and offspring (referred to as CON, RES, and OVER) were euthanized and longissimus dorsi (LM), semitendinosus (STN), and triceps brachii (TB) were collected at each time point for histological and RNA-Seq analysis. In fetuses and offspring, we did not observe an effect of diet on cross-sectional area (CSA), but CSA increased over time (P < 0.05). At day 90, RES and OVER had reduced secondary:primary muscle fiber ratios in LM (P < 0.05), but not in STN and TB. However, in STN and TB percent PAX7-positive cells were decreased compared with CON (P < 0.05). Maternal diet altered LM mRNA expression of 20 genes (7 genes downregulated in OVER and 2 downregulated in RES compared with CON; 5 downregulated in OVER compared with RES; false discovery rate (FDR)-adj. P < 0.05). A diet by time interaction was not observed for any genes in the RNA-Seq analysis; however, 2,205 genes were differentially expressed over time between days 90 and 135 and birth (FDR-adj. P < 0.05). Specifically, consistent with increased protein accretion, changes in muscle function, and increased metabolic activity during myogenesis, changes in genes involved in cell cycle, metabolic processes, and protein synthesis were observed during fetal myogenesis. In conclusion, poor maternal nutrition during gestation contributes to altered offspring muscle growth during early fetal development which persists throughout the fetal stage. Based on muscle-type-specific effects of maternal diet, it is important to evaluate more than one type of muscle to fully elucidate the effects of maternal diet on offspring muscle development.

Maternal Restricted- and Over-Feeding During Gestation Result in Distinct Lipid and Amino Acid Metabolite Profiles in the Longissimus Muscle of the Offspring.

Maternal over- and restricted-feeding during gestation have similar negative consequences for the offspring, including decreased muscularity, increased adiposity, and altered metabolism. Our objective was to determine the effects of poor maternal nutrition during gestation (over- and restricted-feeding) on the offspring muscle metabolite profile. Pregnant ewes (n = 47) were fed 60% (RES), 100% (CON), or 140% (OVER) of NRC requirements starting at day 30.2 ± 0.2 of gestation. Offspring sample collection occurred at days 90 and 135 of gestation, and within 24 h of birth. C2C12 myoblasts were cultured in serum collected from offspring at birth (n = 18; 6 offspring per treatment) for analysis of oxidative and glycolytic capacity. Unbiased metabolite analysis of longissimus muscle samples (n = 72; 8 fetuses per treatment per time point) was performed using mass spectrometry. Data were analyzed by ANOVA for main effects of treatment, time point, and their interaction. Cells cultured in serum from RES offspring exhibited increased proton leak 49% (p = 0.01) compared with CON, but no other variables of mitochondrial respiration or glycolytic function were altered. Mass spectrometry identified 612 metabolites. Principle component analysis identified day of gestation as the primary driver of metabolic change; however, maternal diet also altered the lipid and amino acid profiles in offspring. The abundance of 53 amino acid metabolites and 89 lipid metabolites was altered in RES compared with CON (p ≤ 0.05), including phospholipids, sphingolipids, and ceramides within the lipid metabolism pathway and metabolites involved in glutamate, histidine, and glutathione metabolism. Similarly, abundance of 63 amino acid metabolites and 70 lipid metabolites was altered in OVER compared with CON (p ≤ 0.05). These include metabolites involved in glutamate, histidine, lysine, and tryptophan metabolism and phosphatidylethanolamine, lysophospholipids, and fatty acids involved in lipid metabolism. Further, the amino acid and lipid profiles diverged between RES and OVER, with 69 amino acid and 118 lipid metabolites differing (p ≤ 0.05) between groups. Therefore, maternal diet affects metabolite abundance in offspring longissimus muscle, specifically metabolites involved in lipid and amino metabolism. These changes may impact post-natal skeletal muscle metabolism, possibly altering energy efficiency and long-term health.

Sustained hypoxemia in late gestation potentiates hepatic gluconeogenic gene expression but does not activate glucose production in the ovine fetus.

Fetal hypoxemia is associated with pregnancy conditions that cause an early activation of fetal glucose production. However, the independent role of hypoxemia to activate this pathway is not well understood. We hypothesized that fetal hypoxemia would activate fetal glucose production by decreasing umbilical glucose uptake and increasing counter-regulatory hormone concentrations. We induced hypoxemia for 9 days with maternal tracheal N2 gas insufflation to reduce maternal and fetal arterial Po2 by ~20% (HOX) compared with fetuses from ewes receiving intratracheal compressed air (CON). At 0.9 of gestation, fetal metabolic studies were performed (n = 7 CON, 11 HOX). Umbilical blood flow rates, net fetal oxygen and glucose uptake rates, and fetal arterial plasma glucose concentrations were not different between the two groups. Fetal glucose utilization rates were lower in HOX versus CON fetuses but not different from umbilical glucose uptake rates, demonstrating the absence of endogenous glucose production. In liver tissue, mRNA expression of gluconeogenic genes G6PC (P < 0.01) and PCK1 (P = 0.06) were six- and threefold greater in HOX fetuses versus CON fetuses. Increased fetal norepinephrine and cortisol concentrations and hepatic G6PC and PCK1 expression were inversely related to fetal arterial Po2. These findings support a role for fetal hypoxemia to act with counter-regulatory hormones to potentiate fetal hepatic gluconeogenic gene expression. However, in the absence of decreased net fetal glucose uptake rates and plasma glucose concentrations, hypoxemia-induced gluconeogenic gene activation is not sufficient to activate fetal glucose production.

Differential effects of intrauterine growth restriction and a hypersinsulinemic-isoglycemic clamp on metabolic pathways and insulin action in the fetal liver.

Intrauterine growth-restricted (IUGR) fetal sheep have increased hepatic glucose production (HGP) that is resistant to suppression during a hyperinsulinemic-isoglycemic clamp (insulin clamp). We hypothesized that the IUGR fetal liver would have activation of metabolic and signaling pathways that support HGP and inhibition of insulin-signaling pathways. To test this, we used transcriptomic profiling with liver samples from control (CON) and IUGR fetuses receiving saline or an insulin clamp. The IUGR liver had upregulation of genes associated with gluconeogenesis/glycolysis, transcription factor regulation, and cytokine responses and downregulation of genes associated with cholesterol synthesis, amino acid degradation, and detoxification pathways. During the insulin clamp, genes associated with cholesterol synthesis and innate immune response were upregulated in CON and IUGR. There were 20-fold more genes differentially expressed during the insulin clamp in IUGR versus CON. These genes were associated with proteasome activation and decreased amino acid and lipid catabolism. We found increased TRB3, JUN, MYC, and SGK1 expression and decreased PTPRD expression as molecular targets for increased HGP in IUGR. As candidate genes for resistance to insulin's suppression of HGP, expression of JUN, MYC, and SGK1 increased more during the insulin clamp in CON compared with IUGR. Metabolites were measured with 1H-nuclear magnetic resonance and support increased amino acid concentrations, decreased mitochondria activity and energy state, and increased cell stress in the IUGR liver. These results demonstrate a robust response, beyond suppression of HGP, during the insulin clamp and coordinate responses in glucose, amino acid, and lipid metabolism in the IUGR fetus.

Evaluation of the Nova Vet Meter for sheep-side monitoring of ß-hydroxybutyric acid (BHBA) and description of ewe BHBA during late gestation in three flocks from the Northeastern U.S.

Prevention of metabolic diseases in small ruminants may improve production efficiency and profitability, yet ewes carrying multiples or who are in poor body condition are at increased susceptibility to develop ketosis. This study evaluated the hand-held Nova Vet Meter to accurately detect ß-hydroxybutyric acid (BHBA) concentrations in ewes and determined the percentage of ewes at moderate (0.8 to 1.5 mmol/L BHBA) and greatest (≥1.6 mmol/L BHBA) risk to develop ketosis during late gestation. To validate the Nova Vet Meter, BHBA concentrations of 104 paired blood samples were measured using the Nova Vet Meter and gold-standard laboratory analysis. Receiver operating characteristics were calculated. The accuracy and sensitivity of detecting BHBA concentrations at 0.8 to 1.5 mmol/L were 94.2% and 97.3%, respectively. The accuracy and sensitivity of detecting BHBA concentrations ≥ 1.6 mmol/L were 98.0% and 50.0%, respectively. Ewe body weight (BW), body condition score (BCS), and BHBA of 117 ewes from three flocks were determined weekly during the four weeks before parturition. During the last three weeks of gestation >20% of ewes were identified with moderate risk to develop ketosis. During the last four weeks of gestation, ewes carrying triplets had reduced BCS (P = 0.0002) and increased BHBA concentrations (P < 0.0001) compared with singleton and twin pregnancies. Ewe BHBA did not correlate with lamb birth weight (R2 = 0.003; P = 0.41). In conclusion, the Nova Vet Meter is suitable for sheep-side BHBA monitoring between 0.8 and 1.5 mmol/L, but further testing is necessary to evaluate BHBA readings ≥1.6 mmol/L.

Laryngeal mask airway and transient hypercapnic hyperpnea for video-endoscopic assessment of unilateral laryngeal paralysis in dogs.

OBJECTIVE: To evaluate the ability to assess laryngeal function and to diagnose unilateral laryngeal paralysis (uLP) via airway endoscopy and carbon dioxide (CO2 ) stimulation. STUDY DESIGN: Experimental study. ANIMALS: Six healthy, adult beagles. METHODS: Dogs were anesthetized with sevoflurane and dexmedetomidine. Laryngeal activity was observed via endoscopy placed through a laryngeal mask airway (LMA). The absolute and normalized glottic gap areas (AGGA and NGGA, respectively) and the glottic length (GL) were measured at inspiration and before and after surgically induced uLP. Measurements were obtained at eupnea and during hypercapnic hyperpnea produced by the administration of CO2 . Values for each hemilarynx were also measured. Video recordings were observed by 2 surgeons who scored function as normal or uLP. RESULTS: The AGGA and NGGA increased similarly during CO2 administration in intact dogs and in dogs with uLP; the GL increased in dogs with uLP but not in intact dogs. The AGGA and NGGA of the intact hemilarynx increased more than those of the affected hemilarynx in dogs with uLP. uLP was correctly identified more frequently by observers at hypercapnic hyperpnea than during eupnea. CONCLUSION: The increase in AGGA and NGGA at peak inspiration during CO2 administration was not limited by uLP, but asymmetry in hemilarynx AGGA and NGGA was observed in dogs with uLP. CO2 administration facilitated the identification of uLP. CLINICAL SIGNIFICANCE: Laryngeal endoscopy through an LMA coupled with administration of CO2 in anesthetized dogs facilitates the observation of arytenoid function and may improve the diagnosis of naturally occurring mild laryngeal paralysis.