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.

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.

Effect of frequent interruptions of sedentary time on nutrient metabolism in sedentary overweight male and female adults.

This study compared 24-h nutrient oxidation responses between a sedentary condition (SED) and a condition in which short 5-min bouts of moderate-intensity physical activity were performed hourly for nine consecutive hours over 4 days (MICRO). To determine whether any shifts in fuel use were due solely to increases in energy expenditure, we also studied a condition consisting of a single isoenergetic 45-min bout of moderate-intensity exercise (ONE). Twenty sedentary overweight or obese adults (10 men/10 women; 32.4 ± 6.3 yr; BMI, 30.6 ± 2.9 kg/m2) completed all three conditions (MICRO, SED, and ONE) in a randomized order. Each condition consisted of a 3-day free-living run-in followed by a 24-h stay in a whole-room calorimeter to measure total energy expenditure (TEE) and substrate utilization. Dietary fat oxidation was also assessed during the chamber stay by administering a [1-13C] oleic acid tracer at breakfast. Energy intake was matched across conditions. Both MICRO and ONE increased TEE relative to SED, resulting in a negative energy balance. HOMA-IR improved in both activity conditions. MICRO increased 24-h carbohydrate oxidation compared with both ONE and SED ( P < 0.01 for both). ONE was associated with higher 24-h total fat oxidation compared with SED, and higher 24-h dietary fat oxidation compared with both SED and MICRO. Differences in substrate oxidation remained significant after adjusting for energy balance. In overweight and obese men and women, breaking up sitting time increased reliance upon carbohydrate as fuel over 24 h, while a single energy-matched continuous bout of exercise preferentially relies upon fat over 24 h. NEW & NOTEWORTHY Insulin sensitivity, as assessed by HOMA-IR, was improved after 4 days of physical activity, independent of frequency and duration of activity bouts. Temporal patterns of activity across the day differentially affect substrate oxidation. Frequent interruptions of sedentary time with short bouts of walking primarily increase 24-h carbohydrate oxidation, whereas an energy-matched single continuous bout of moderate intensity walking primarily increased 24-h fat oxidation.

Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth-restricted fetal sheep.

KEY POINTS: Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass, which may contribute to insulin resistance and the development of diabetes. We demonstrate slower hindlimb linear growth and muscle protein synthesis rates that match the reduced hindlimb blood flow and oxygen consumption rates in IUGR fetal sheep. These adaptations resulted in hindlimb blood flow rates in IUGR that were similar to control fetuses on a weight-specific basis. Net hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was significantly lower in IUGR fetal sheep. Among all fetuses, blood O2 saturation and plasma glucose, insulin and insulin-like growth factor-1 were positively associated and norepinephrine was negatively associated with hindlimb weight. These results further our understanding of the metabolic and hormonal adaptations to reduced oxygen and nutrient supply with placental insufficiency that develop to slow hindlimb growth and muscle protein accretion. ABSTRACT: Reduced skeletal muscle mass in the fetus with intrauterine growth restriction (IUGR) persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake and protein accretion rates in skeletal muscle, late gestation control (CON) (n = 8) and IUGR (n = 13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate and fractional synthetic rate were lower in IUGR compared to CON (P < 0.05). Absolute hindlimb blood flow was reduced in IUGR (IUGR: 32.9 ± 5.6 ml min-1 ; CON: 60.9 ± 6.5 ml min-1 ; P < 0.005), although flow normalized to hindlimb weight was similar between groups. Hindlimb oxygen consumption rate was lower in IUGR (IUGR: 10.4 ± 1.4 µmol min-1  100 g-1 ; CON: 14.7 ± 1.3 µmol min-1  100 g-1 ; P < 0.05). Hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was lower in IUGR (IUGR: 1.3 ± 0.5 µmol min-1  100 g-1 ; CON: 2.9 ± 0.2 µmol min-1  100 g-1 ; P < 0.05). Blood O2 saturation (r2  = 0.80, P < 0.0001) and plasma glucose (r2  = 0.68, P < 0.0001), insulin (r2  = 0.40, P < 0.005) and insulin-like growth factor (IGF)-1 (r2  = 0.80, P < 0.0001) were positively associated and norepinephrine (r2  = 0.59, P < 0.0001) was negatively associated with hindlimb weight. Slower hindlimb linear growth and muscle protein synthesis rates match reduced hindlimb blood flow and oxygen consumption rates in the IUGR fetus. Metabolic adaptations to slow hindlimb growth are probably hormonally-mediated by mechanisms that include increased fetal norepinephrine and reduced IGF-1 and insulin.

The beneficial effects of exercise on cartilage are lost in mice with reduced levels of ECSOD in tissues.

Osteoarthritis (OA) is associated with increased mechanical damage to joint cartilage. We have previously found that extracellular superoxide dismutase (ECSOD) is decreased in OA joint fluid and cartilage, suggesting oxidant damage may play a role in OA. We explored the effect of forced running as a surrogate for mechanical damage in a transgenic mouse with reduced ECSOD tissue binding. Transgenic mice heterozygous (Het) for the human ECSOD R213G polymorphism and 129-SvEv (wild-type, WT) mice were exposed to forced running on a treadmill for 45 min/day, 5 days/wk, over 8 wk. At the end of the running protocol, knee joint tissue was obtained for histology, immunohistochemistry, and protein analysis. Sedentary Het and WT mice were maintained for comparison. Whole tibias were studied for bone morphometry, finite element analysis, and mechanical testing. Forced running improved joint histology in WT mice. However, when ECSOD levels were reduced, this beneficial effect with running was lost. Het ECSOD runner mice had significantly worse histology scores compared with WT runner mice. Runner mice for both strains had increased bone strength in response to the running protocol, while Het mice showed evidence of a less robust bone structure in both runners and untrained mice. Reduced levels of ECSOD in cartilage produced joint damage when joints were stressed by forced running. The bone tissues responded to increased loading with hypertrophy, regardless of mouse strain. We conclude that ECSOD plays an important role in protecting cartilage from damage caused by mechanical loading.

A common polymorphism in extracellular superoxide dismutase affects cardiopulmonary disease risk by altering protein distribution.

BACKGROUND: The enzyme extracellular superoxide dismutase (EC-SOD; SOD3) is a major antioxidant defense in lung and vasculature. A nonsynonomous single-nucleotide polymorphism in EC-SOD (rs1799895) leads to an arginine to glycine amino acid substitution at position 213 (R213G) in the heparin-binding domain. In recent human genetic association studies, this single-nucleotide polymorphism attenuates the risk of lung disease, yet paradoxically increases the risk of cardiovascular disease. METHODS AND RESULTS: Capitalizing on the complete sequence homology between human and mouse in the heparin-binding domain, we created an analogous R213G single-nucleotide polymorphism knockin mouse. The R213G single-nucleotide polymorphism did not change enzyme activity, but shifted the distribution of EC-SOD from lung and vascular tissue to extracellular fluid (eg, bronchoalveolar lavage fluid and plasma). This shift reduces susceptibility to lung disease (lipopolysaccharide-induced lung injury) and increases susceptibility to cardiopulmonary disease (chronic hypoxic pulmonary hypertension). CONCLUSIONS: We conclude that EC-SOD provides optimal protection when localized to the compartment subjected to extracellular oxidative stress: thus, the redistribution of EC-SOD from the lung and pulmonary circulation to the extracellular fluids is beneficial in alveolar lung disease but detrimental in pulmonary vascular disease. These findings account for the discrepant risk associated with R213G in humans with lung diseases compared with cardiovascular diseases.

The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the naturally occurring R213G substitution.

Extracellular superoxide dismutase (EC-SOD) is responsible for the dismutation of the superoxide radical produced in the extracellular space and known to be expressed by inflammatory cells, including macrophages and neutrophils. Here we show that EC-SOD is produced by resting macrophages and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand-binding capacity, this could not be correlated to proteolytic removal of the ECM-binding region, because the integrity of the material recovered from the medium was comparable to that of the cell surface-associated protein. The naturally occurring R213G amino acid substitution located in the ECM-binding region of EC-SOD is known to affect the binding characteristics of the protein. However, the analysis of macrophages expressing R213G EC-SOD did not present evidence of an altered cellular distribution. Our results suggest that EC-SOD plays a dynamic role in the inflammatory response mounted by activated macrophages.

Importance of manual validation for the identification of phosphopeptides using a linear ion trap mass spectrometer.

Accurate determination of protein phosphorylation is challenging, particularly for researchers who lack access to a high-accuracy mass spectrometer. In this study, multiple protocols were used to enrich phosphopeptides, and a rigorous filtering workflow was used to analyze the resulting samples. Phosphopeptides were enriched from cultured rat renal proximal tubule cells using three commonly used protocols and a dual method that combines separate immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO(2)) chromatography, termed dual IMAC (DIMAC). Phosphopeptides from all four enrichment strategies were analyzed by liquid chromatography-multiple levels of mass spectrometry (LC-MS(n)) neutral-loss scanning using a linear ion trap mass spectrometer. Initially, the resulting MS(2) and MS(3) spectra were analyzed using PeptideProphet and database search engine thresholds that produced a false discovery rate (FDR) of <1.5% when searched against a reverse database. However, only 40% of the potential phosphopeptides were confirmed by manual validation. The combined analyses yielded 110 confidently identified phosphopeptides. Using less-stringent initial filtering thresholds (FDR of 7-9%), followed by rigorous manual validation, 262 unique phosphopeptides, including 111 novel phosphorylation sites, were identified confidently. Thus, traditional methods of data filtering within widely accepted FDRs were inadequate for the analysis of low-resolution phosphopeptide spectra. However, the combination of a streamlined front-end enrichment strategy and rigorous manual spectral validation allowed for confident phosphopeptide identifications from a complex sample using a low-resolution ion trap mass spectrometer.

TFIIA plays a role in the response to oxidative stress.

To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.