Deletion of miPEP in adipocytes protects against obesity and insulin resistance by boosting muscle metabolism.

Mitochondria facilitate thousands of biochemical reactions, covering a broad spectrum of anabolic and catabolic processes. Here we demonstrate that the adipocyte mitochondrial proteome is markedly altered across multiple models of insulin resistance and reveal a consistent decrease in the level of the mitochondrial processing peptidase miPEP. To experimentally test this observation, we generated adipocyte-specific miPEP knockout mice to interrogate its role in the aetiology of insulin resistance. We observed a strong phenotype characterised by enhanced insulin sensitivity and reduced adiposity, despite normal food intake and physical activity. Strikingly, these phenotypes vanished when mice were housed at thermoneutrality, suggesting that metabolic protection conferred by miPEP deletion hinges upon a thermoregulatory process. Tissue specific analysis of miPEP deficient mice revealed an increment in muscle metabolism, and upregulation of the protein FBP2 that is involved in ATP hydrolysis in the gluconeogenic pathway. These findings suggest that miPEP deletion initiates a compensatory increase in skeletal muscle metabolism acting as a protective mechanism against diet-induced obesity and insulin resistance.

The metabolic consequences of 'yo-yo' dieting are markedly influenced by genetic diversity.

BACKGROUND: Weight loss can improve the metabolic complications of obesity. However, it is unclear whether insulin resistance persists despite weight loss and whether any protective benefits are preserved following weight regain (weight cycling). The impact of genetic background on weight cycling is undocumented. We aimed to investigate the effects of weight loss and weight cycling on metabolic outcomes and sought to clarify the role of genetics in this relationship. METHOD: Both C57BL/6 J and genetically heterogeneous Diversity Outbred Australia (DOz) mice were alternately fed high fat Western-style diet (WD) and a chow diet at 8-week intervals. Metabolic measures including body composition, glucose tolerance, pancreatic beta cell activity, liver lipid levels and adipose tissue insulin sensitivity were determined. RESULTS: After diet switch from WD (8-week) to chow (8-week), C57BL/6 J mice displayed a rapid normalisation of body weight, adiposity, hyperinsulinemia, liver lipid levels and glucose uptake into adipose tissue comparable to chow-fed controls. In response to the same dietary intervention, genetically diverse DOz mice conversely maintained significantly higher fat mass and insulin levels compared to chow-fed controls and exhibited much more profound interindividual variability than C57BL/6 J mice. Weight cycled (WC) animals were re-exposed to WD (8-week) and compared to age-matched controls fed 8-week WD for the first time (LOb). In C57BL/6 J but not DOz mice, WC animals had significantly higher blood insulin levels than LOb controls. All WC animals exhibited significantly greater beta cell activity than LOb controls despite similar fat mass, glucose tolerance, liver lipid levels and insulin-stimulated glucose uptake in adipose tissue. CONCLUSION: Following weight loss, metabolic outcomes return to baseline in C57BL/6 J mice with obesity. However, genetic diversity significantly impacts this response. A period of weight loss does not provide lasting benefits after weight regain, and weight cycling is detrimental and associated with hyperinsulinemia and elevated basal insulin secretion.

The genetic and dietary landscape of the muscle insulin signalling network.

Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.

When we eat, the pancreas releases a hormone called insulin, which helps our tissues absorb glucose. Insulin works by triggering a cascade of events in cells, which include adding chemical tags called phosphate groups at thousands of specific locations on proteins. This tag causes the changes needed to move glucose from the blood into cells and also regulates many other essential functions in the cell. If this process stops working and the body becomes resistant to the effects of insulin, it can lead to type 2 diabetes. This can result from a complex combination of genetic and lifestyle factors, which are difficult to study systematically in people. An alternative approach to understand these influences is to study mice, which are commonly used to investigate metabolic diseases and have contributed to our understanding of the mechanisms of type 2 diabetes. Using carefully bred mice allows precise control of their genetics and environment, revealing the independent and joint effects of these factors. Monitoring differences in the phosphate groups on proteins, van Gerwen et al. studied five distinct inbred mouse strains fed either an ordinary diet or one that was high in fat and sugar. Nearly half of the biochemical events triggered by insulin were altered by genetics on the ordinary diet. High-fat, high-sugar feeding also reshaped the pattern of phosphate tags depending on the mouse strain. By examining these cellular responses, van Gerwen et al. identified proteins that may regulate the insulin response in muscle cells. Increasing the activity of one of these enzymes reversed insulin resistance in skeletal muscle cells grown in the laboratory. This research underscores the importance of genetics in controlling insulin responses and shaping the impact of environmental challenges. It establishes a new opportunity in personalised medicine, which seeks to understand how an individual's genetics combine with their lifestyle to shape health. Furthermore, it identifies potential new targets for treating insulin resistance, paving the way for future research to develop more effective diabetes treatments.

Dual Tracer Test to Measure Tissue-Specific Insulin Action in Individual Mice Identifies In Vivo Insulin Resistance Without Fasting Hyperinsulinemia.

The ability of metabolically active tissues to increase glucose uptake in response to insulin is critical to whole-body glucose homeostasis. This report describes the Dual Tracer Test, a robust method involving sequential retro-orbital injection of [14C]2-deoxyglucose ([14C]2DG) alone, followed 40 min later by injection of [3H]2DG with a maximal dose of insulin to quantify both basal and insulin-stimulated 2DG uptake in the same mouse. The collection of both basal and insulin-stimulated measures from a single animal is imperative for generating high-quality data since differences in insulin action may be misinterpreted mechanistically if basal glucose uptake is not accounted for. The approach was validated in a classic diet-induced model of insulin resistance and a novel transgenic mouse with reduced GLUT4 expression that, despite ubiquitous peripheral insulin resistance, did not exhibit fasting hyperinsulinemia. This suggests that reduced insulin-stimulated glucose disposal is not a primary contributor to chronic hyperinsulinemia. The Dual Tracer Test offers a technically simple assay that enables the study of insulin action in many tissues simultaneously. By administering two tracers and accounting for both basal and insulin-stimulated glucose transport, this assay halves the required sample size for studies in inbred mice and demonstrates increased statistical power to detect insulin resistance, relative to other established approaches, using a single tracer. The Dual Tracer Test is a valuable addition to the metabolic phenotyping toolbox.

Leveraging genetic diversity to identify small molecules that reverse mouse skeletal muscle insulin resistance.

Systems genetics has begun to tackle the complexity of insulin resistance by capitalising on computational advances to study high-diversity populations. 'Diversity Outbred in Australia (DOz)' is a population of genetically unique mice with profound metabolic heterogeneity. We leveraged this variance to explore skeletal muscle's contribution to whole-body insulin action through metabolic phenotyping and skeletal muscle proteomics of 215 DOz mice. Linear modelling identified 553 proteins that associated with whole-body insulin sensitivity (Matsuda Index) including regulators of endocytosis and muscle proteostasis. To enrich for causality, we refined this network by focusing on negatively associated, genetically regulated proteins, resulting in a 76-protein fingerprint of insulin resistance. We sought to perturb this network and restore insulin action with small molecules by integrating the Broad Institute Connectivity Map platform and in vitro assays of insulin action using the Prestwick chemical library. These complementary approaches identified the antibiotic thiostrepton as an insulin resistance reversal agent. Subsequent validation in ex vivo insulin-resistant mouse muscle and palmitate-induced insulin-resistant myotubes demonstrated potent insulin action restoration, potentially via upregulation of glycolysis. This work demonstrates the value of a drug-centric framework to validate systems-level analysis by identifying potential therapeutics for insulin resistance.

A role for ß-catenin in diet-induced skeletal muscle insulin resistance.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. ß-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle ß-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated ß-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific ß-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower ß-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated ß-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, ß-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total ß-catenin expression was unchanged. These findings suggest that ß-catenin dysfunction is associated with the development of insulin resistance.

Systems-level analysis of insulin action in mouse strains provides insight into tissue- and pathway-specific interactions that drive insulin resistance.

Skeletal muscle and adipose tissue insulin resistance are major drivers of metabolic disease. To uncover pathways involved in insulin resistance, specifically in these tissues, we leveraged the metabolic diversity of different dietary exposures and discrete inbred mouse strains. This revealed that muscle insulin resistance was driven by gene-by-environment interactions and was strongly correlated with hyperinsulinemia and decreased levels of ten key glycolytic enzymes. Remarkably, there was no relationship between muscle and adipose tissue insulin action. Adipocyte size profoundly varied across strains and diets, and this was strongly correlated with adipose tissue insulin resistance. The A/J strain, in particular, exhibited marked adipocyte insulin resistance and hypertrophy despite robust muscle insulin responsiveness, challenging the role of adipocyte hypertrophy per se in systemic insulin resistance. These data demonstrate that muscle and adipose tissue insulin resistance can occur independently and underscore the need for tissue-specific interrogation to understand metabolic disease.

α1-Antitrypsin A treatment attenuates neutrophil elastase accumulation and enhances insulin sensitivity in adipose tissue of mice fed a high-fat diet.

Neutrophils accumulate in insulin-sensitive tissues during obesity and may play a role in impairing insulin sensitivity. The major serine protease expressed by neutrophils is neutrophil elastase (NE), which is inhibited endogenously by α1-antitrypsin A (A1AT). We investigated the effect of exogenous (A1AT) treatment on diet-induced metabolic dysfunction. Male C57Bl/6j mice fed a chow or a high-fat diet (HFD) were randomized to receive intraperitoneal injections three times weekly of either Prolastin (human A1AT; 2 mg) or vehicle (PBS) for 10 wk. Prolastin treatment did not affect plasma NE concentration, body weight, glucose tolerance, or insulin sensitivity in chow-fed mice. In contrast, Prolastin treatment attenuated HFD-induced increases in plasma and white adipose tissue (WAT) NE without affecting circulatory neutrophil levels or increases in body weight. Prolastin-treated mice fed a HFD had improved insulin sensitivity, as assessed by insulin tolerance test, and this was associated with higher insulin-dependent IRS-1 (insulin receptor substrate) and AktSer473 phosphorylation, and reduced inflammation markers in WAT but not liver or muscle. In 3T3-L1 adipocytes, Prolastin reversed recombinant NE-induced impairment of insulin-stimulated glucose uptake and IRS-1 phosphorylation. Furthermore, PDGF mediated p-AktSer473 activation and glucose uptake (which is independent of IRS-1) was not affected by recombinant NE treatment. Collectively, our findings suggest that NE infiltration of WAT during metabolic overload contributes to insulin resistance by impairing insulin-induced IRS-1 signaling.NEW & NOTEWORTHY Neutrophils accumulate in peripheral tissues during obesity and are critical coordinators of tissue inflammatory responses. Here, we provide evidence that inhibition of the primary neutrophil protease, neutrophil elastase, with α1-antitrypsin A (A1AT) can improve insulin sensitivity and glucose homeostasis of mice fed a high-fat diet. This was attributed to improved insulin-induced IRS-1 phosphorylation in white adipose tissue and provides further support for a role of neutrophils in mediating diet-induced peripheral tissue insulin resistance.

ß-Catenin is required for optimal exercise- and contraction-stimulated skeletal muscle glucose uptake.

KEY POINTS: Loss of ß-catenin impairs in vivo and isolated muscle exercise/contraction-stimulated glucose uptake. ß-Catenin is required for exercise-induced skeletal muscle actin cytoskeleton remodelling. ß-Catenin675 phosphorylation during exercise may be intensity dependent. ABSTRACT: The conserved structural protein ß-catenin is an emerging regulator of vesicle trafficking in multiple tissues and supports insulin-stimulated glucose transporter 4 (GLUT4) translocation in skeletal muscle by facilitating cortical actin remodelling. Actin remodelling may be a convergence point between insulin and exercise/contraction-stimulated glucose uptake. Here we investigated whether ß-catenin is involved in regulating exercise/contraction-stimulated glucose uptake. We report that the muscle-specific deletion of ß-catenin induced in adult mice (BCAT-mKO) impairs both exercise- and contraction (isolated muscle)-induced glucose uptake without affecting running performance or canonical exercise signalling pathways. Furthermore, high intensity exercise in mice and contraction of myotubes and isolated muscles led to the phosphorylation of ß-cateninS675 , and this was impaired by Rac1 inhibition. Moderate intensity exercise in control and Rac1 muscle-specific knockout mice did not induce muscle ß-cateninS675 phosphorylation, suggesting exercise intensity-dependent regulation of ß-cateninS675 . Introduction of a non-phosphorylatable S675A mutant of ß-catenin into myoblasts impaired GLUT4 translocation and actin remodelling stimulated by carbachol, a Rac1 and RhoA activator. Exercise-induced increases in cross-sectional phalloidin staining (F-actin marker) of gastrocnemius muscle was impaired in muscle from BCAT-mKO mice. Collectively our findings suggest that ß-catenin is required for optimal glucose transport in muscle during exercise/contraction, potentially via facilitating actin cytoskeleton remodelling.

Sirtuin 1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle.

While it has long been known that contraction robustly stimulates skeletal muscle glucose uptake, the molecular steps regulating this increase remain incompletely defined. The mammalian ortholog of Sir2, sirtuin 1 (SIRT1), is an NAD+-dependent protein deacetylase that is thought to link perturbations in energy flux associated with exercise to subsequent cellular adaptations. Nevertheless, its role in contraction-stimulated glucose uptake has not been described. The objective of this study was to determine the importance of SIRT1 to contraction-stimulated glucose uptake in mouse skeletal muscle. Using a radioactive 2-deoxyglucose uptake (2DOGU) approach, we measured ex vivo glucose uptake in unstimulated (rested) and electrically stimulated (100 Hz contraction every 15 s for 10 min; contracted) extensor digitorum longus (EDL) and soleus from ∼15-wk-old male and female mice with muscle-specific knockout of SIRT1 deacetylase activity and their wild-type littermates. Skeletal muscle force decreased over the contraction protocol, although there were no differences in the rate of fatigue between genotypes. In EDL and soleus, loss of SIRT1 deacetylase activity did not affect contraction-induced increase in glucose uptake in either sex. Interestingly, the absolute rate of contraction-stimulated 2DOGU was ∼1.4-fold higher in female compared with male mice, regardless of muscle type. Taken together, our findings demonstrate that SIRT1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle. Moreover, to our knowledge, this is the first demonstration of sex-based differences in contraction-stimulated glucose uptake in mouse skeletal muscle.NEW & NOTEWORTHY Here, we demonstrate that glucose uptake in response to ex vivo contractions is not affected by the loss of sirtuin 1 (SIRT1) deacetylase function in muscle, regardless of sex or muscle type. Interestingly, however, similar to studies on insulin-stimulated glucose uptake, we demonstrate that contraction-stimulated glucose uptake is robustly higher in female compared with the male skeletal muscle. To our knowledge, this is the first demonstration of sex-based differences in contraction-stimulated glucose uptake in skeletal muscle.

ß-catenin regulates muscle glucose transport via actin remodelling and M-cadherin binding.

OBJECTIVE: Skeletal muscle glucose disposal following a meal is mediated through insulin-stimulated movement of the GLUT4-containing vesicles to the cell surface. The highly conserved scaffold-protein ß-catenin is an emerging regulator of vesicle trafficking in other tissues. Here, we investigated the involvement of ß-catenin in skeletal muscle insulin-stimulated glucose transport. METHODS: Glucose homeostasis and transport was investigated in inducible muscle specific ß-catenin knockout (BCAT-mKO) mice. The effect of ß-catenin deletion and mutation of ß-catenin serine 552 on signal transduction, glucose uptake and protein-protein interactions were determined in L6-G4-myc cells, and ß-catenin insulin-responsive binding partners were identified via immunoprecipitation coupled to label-free proteomics. RESULTS: Skeletal muscle specific deletion of ß-catenin impaired whole-body insulin sensitivity and insulin-stimulated glucose uptake into muscle independent of canonical Wnt signalling. In response to insulin, ß-catenin was phosphorylated at serine 552 in an Akt-dependent manner, and in L6-G4-myc cells, mutation of ß-cateninS552 impaired insulin-induced actin-polymerisation, resulting in attenuated insulin-induced glucose transport and GLUT4 translocation. ß-catenin was found to interact with M-cadherin in an insulin-dependent ß-cateninS552-phosphorylation dependent manner, and loss of M-cadherin in L6-G4-myc cells attenuated insulin-induced actin-polymerisation and glucose transport. CONCLUSIONS: Our data suggest that ß-catenin is a novel mediator of glucose transport in skeletal muscle and may contribute to insulin-induced actin-cytoskeleton remodelling to support GLUT4 translocation.

Prolonged treatment with a PI3K p110α inhibitor causes sex- and tissue-dependent changes in antioxidant content, but does not affect mitochondrial function.

Genetic inhibition of the p110α isoform of phosphatidylinositol-3-kinase (PI3K) can increase murine lifespan, enhance mitochondrial function and alter tissue-specific oxidative balance. Here, we investigated whether pharmacological inhibition of the p110α isoform of PI3K induces similar enhancement of mitochondrial function in middle-aged mice. Eight-month-old male and female mice were fed a diet containing 0.3 g/kg of the p110α-selective inhibitor BYL-719 (BYL) or a vehicle diet (VEH) for 6 weeks. Mice consuming BYL-719 had higher blood glucose and insulin, and tended towards decreased body weight. After 72 h, gene expression of the mitochondrial biogenesis mediators Pgc1α, Tfam and Nrf1 was greater in liver of BYL-719 males only, but unchanged in skeletal muscle of either sex. Six weeks of BYL-719 treatment did not affect mitochondrial content or function in the liver or skeletal muscle of either sex. In livers of males only, the expression of the antioxidant genes Nfe2l2, Cat, Sod1 and Sod2 increased within 72 h of BYL-719 treatment, and remained higher after 6 weeks. This was associated with an increase in hepatic GSH content and catalase protein expression, and lower H2O2 levels. Our results suggest that pharmacological inhibition of p110α in adult mice does not affect liver or skeletal muscle mitochondrial function, but does show sex- and tissue-specific effects on up-regulation of antioxidant response.

Effects of zinc source and dietary concentration on serum zinc concentrations, growth performance, wool and reproductive characteristics in developing rams.

Dietary Zn has significant impacts on the growth and development of breeding rams. The objectives of this study were to evaluate the effects of dietary Zn source and concentration on serum Zn concentration, growth performance, wool traits and reproductive performance in rams. Forty-four Targhee rams (14 months; 68 ± 18 kg BW) were used in an 84-day completely randomized design and were fed one of three pelleted dietary treatments: (1) a control without fortified Zn (CON; n = 15; ~1 × NRC); (2) a diet fortified with a Zn amino acid complex (ZnAA; n = 14; ~2 × NRC) and (3) a diet fortified with ZnSO4 (ZnSO4; n = 15; ~2 × NRC). Growth and wool characteristics measured throughout the course of the study were BW, average daily gain (ADG), dry matter intake (DMI), feed efficiency (G : F), longissimus dorsi muscle depth (LMD), back fat (BF), wool staple length (SL) and average fibre diameter (AFD). Blood was collected from each ram at four time periods to quantify serum Zn and testosterone concentrations. Semen was collected 1 to 2 days after the trial was completed. There were no differences in BW (P = 0.45), DMI (P = 0.18), LMD (P = 0.48), BF (P = 0.47) and AFD (P = 0.9) among treatment groups. ZnSO4 had greater (P ≤ 0.03) serum Zn concentrations compared with ZnAA and CON treatments. Rams consuming ZnAA had greater (P ≤ 0.03) ADG than ZnSO4 and CON. There tended to be differences among groups for G : F (P = 0.06), with ZnAA being numerically greater than ZnSO4 and CON. Wool staple length regrowth was greater (P < 0.001) in ZnSO4 and tended to be longer (P = 0.06) in ZnAA treatment group compared with CON. No differences were observed among treatments in scrotal circumference, testosterone, spermatozoa concentration within ram semen, % motility, % live sperm and % sperm abnormalities (P ≥ 0.23). Results indicated beneficial effects of feeding increased Zn concentrations to developing Targhee rams, although Zn source elicited differential responses in performance characteristics measured.

Effects of late gestation shearing on BW, feed intake and plasma metabolite concentrations in Rambouillet ewes managed outdoors during winter.

The majority of lambs in the United States are born from late winter to early spring and pregnant ewes are generally sheared in the last third of pregnancy. Although there are benefits to shearing before parturition, shorn animals may be more vulnerable to the cold, highly variable climatic conditions associated with these seasons. The objective of this study was to determine if late gestation shearing induces differences in individual BW, dry matter intake (DMI) and plasma metabolite concentration of finewool ewes managed outdoors during winter. Thirty-six mature, pregnant Rambouillet ewes (3.8±0.45 years; 76.8±11.4 kg) were managed in a drylot with ad libitum access to pelleted alfalfa in bunks capable of measuring individual daily DMI. The treatment group consisted of ewes sheared at ~5 weeks before the estimated parturition date (shorn; n=18). Unshorn ewes (n=18) remained in full fleece throughout the experiment and were shorn on the last day of the experiment ~2 weeks before the estimated parturition date. Blood was collected on days 0 (before shearing shorn group), 7, 14 and 21 (before shearing unshorn group) of the trial, and plasma was isolated and analyzed for non-esterified fatty acid (NEFA), ß-hydroxybutyrate (BHB) and glucose (GLU) concentrations. There was no effect of shearing on ewe DMI or BW during the trial (P⩾0.35). Plasma NEFA and GLU concentrations were similar (P⩾0.36) between shearing groups, though plasma BHB concentration was 103.7 µmol/l greater (24.1%; P<0.01) in unshorn ewes. Lamb BW at birth was not affected (P=0.30) by ewe shearing treatment. Under conditions of this study, no differences in economically important aspects of sheep production were observed between shorn and unshorn pregnant ewes.

Using clinical and genetic data to predict pulmonary hypertension in bronchopulmonary dysplasia.

AIM: Pulmonary hypertension significantly increases morbidity and mortality in infants with bronchopulmonary dysplasia. The frequency of single nucleotide polymorphisms in arginase-1 (ARG1 rs2781666) and dimethylarginine dimethylaminohydrolase-1 (DDAH1 rs480414) genes has been found to differ in a cohort of bronchopulmonary dysplasia patients with pulmonary hypertension (cases) and without pulmonary hypertension (controls). Therefore, we tested the hypothesis that combining these genotypes with phenotypic data would better predict pulmonary hypertension in bronchopulmonary dysplasia patients. METHODS: Bronchopulmonary dysplasia patients (n = 79) born at <35 weeks gestation were studied. Pulmonary hypertension was diagnosed by echocardiographic criteria (n = 20). ROC curves to predict pulmonary hypertension in bronchopulmonary dysplasia were generated from genotype and/or clinical data. RESULTS: Cases were born at an earlier gestation and weighed less at birth than did controls. ROC curves for rs2781666 had an AUC of 0.61, while rs480414 had an AUC of 0.66. Together, the AUC was 0.70. When clinical data were added to the genetic model, AUC was 0.73. CONCLUSION: These findings demonstrate that ROC predictive modelling of pulmonary hypertension in bronchopulmonary dysplasia improves with inclusion of both genotypic and phenotypic data. Further refinement of these types of models could facilitate the implementation of precision medicine approaches to pulmonary hypertension in bronchopulmonary dysplasia.

Substituting ground woody plants for cottonseed hulls in lamb feedlot diets: Growth performance, blood serum chemistry, and rumen fluid parameters.

Effects of using ground woody plants in Rambouillet wether lamb feedlot diets on growth performance, blood serum, and rumen parameters were evaluated. A randomized design study was used with 2 feeding periods (70% concentrate diet from d 0 to 27 [Period 1] and 86% concentrate diet from d 28 to 57 [Period 2]); the concentrate portion of the diets mainly consisted of dried distiller's grains with solubles and sorghum grain. Lambs were individually fed 6 diets that differed only by roughage source ( = 8 animals/treatment; 32.9 ± 3.2 kg initial BW): either cottonseed hulls (CSH; control) or ground wood consisting of redberry juniper (RED), blueberry juniper (BLUE), one-seeded juniper (ONE), or eastern red cedar (ERC) spp. or honey mesquite (MESQ; ). Using ground wood vs. CSH as the roughage source did not affect ( > 0.12) BW. There tended to be a treatment × day interaction ( = 0.07) for lamb DMI, attributed to Period 1 when DMI was greater ( < 0.05) for lambs fed CSH vs. RED, ONE, ERC, or MESQ during the first 14 d and greater ( < 0.05) during d 14 to 28 vs. lambs fed ERC. Overall, ADG was less for lambs fed ERC ( < 0.10) or MESQ ( < 0.05) vs. lambs fed CSH, but G:F was similar ( > 0.10) among all lambs. Dietary treatments did not affect ( > 0.15) ruminal pH, but treatment × day interactions ( < 0.05) were observed for rumen fluid ammonia N or molar proportions of propionate and butyrate; few differences were observed within day. However, overall, lambs fed RED or MESQ had greater ( < 0.05) total rumen VFA than lambs fed CSH. A treatment × day interaction ( = 0.04) was observed for the acetate:propionate ratio, but no differences ( > 0.10) were observed within day. Treatment × day interactions ( < 0.05) were observed for blood serum glucose, γ-glutamyl transferase, the albumin:globulin ratio, total bilirubin, ß-hydroxybutyrate, P, Cl, and Mg, with most results being less for lambs fed the wood-based diets than for lambs fed the CSH diets. Results suggested that even though lamb DMI was reduced during the growing period when diets contained 30% ground woody products (RED, ONE, ERC, and MESQ), animal health and rumen fluid parameters were not negatively affected by ground woody plants.

Effects of feeding ground redberry juniper (Juniperus pinchotii) to gestating ewes on pre- and postpartum performance, serum metabolites and hormones, milk fatty acid composition, and progeny preweaning performance.

The objective of this research was to evaluate effects of replacing sorghum × Sudangrass hay with ground juniper in gestating ewe supplements on pre- and postpartum growth performance, serum metabolites and hormonal concentrations, milk fatty acid composition, and progeny preweaning performance. In a completely randomized design, commercial Rambouillet ewes (age = 3 to 5 yr; initial BW = 65.2 ± 1.6 kg) on a base diet of long-stem sorghum × Sudangrass hay were assigned to 1 of 4 dietary supplements in which ground juniper replaced 0% (CNTL), 33% (18JUN), 66% (36JUN), or 100% (54JUN) of the ground sorghum × Sudangrass hay in a pelleted supplement with ground juniper from d 38 ± 4 of gestation to 2 d postpartum. Treatment DM diet intake overall (g/kg BW) in ewes receiving no juniper was similar ( ≥ 0.38) to that of those receiving increasing concentrations of juniper. Changes in ewe BW and BCS were similar ( ≥ 0.24) in ewes throughout gestation. All serum metabolites and hormones were within normal clinical ranges; however, serum IGF-1 decreased linearly ( = 0.003), alanine increased (linear; = 0.003), and serum Na decreased (linear; = 0.049) as the percentage of juniper increased in the diet. Ewe milk fatty acid composition was similar ( > 0.05) for the majority of fatty acids across treatment groups, with the exception of arachidonic acid (C20:4n6) being greater ( < 0.02) in 54JUN vs. CNTL ewe milk. Lamb birth weights were similar ( = 0.13), whereas lamb ADG tended to differ (quadratic; = 0.06) from d 0 to 14, with 18JUN being the least. At weaning, BW tended ( = 0.09) to linearly decrease in lambs born to ewes consuming greater concentrations of juniper but were not different ( = 0.26) between CNTL and 18JUN, 36JUN, and 54JUN. Results indicated that feeding increasing levels of ground juniper in supplements did not negatively alter ewe performance or serum metabolites and hormones measured pre- and postpartum. Lamb birth weight and preweaning performance appeared unaffected by maternal consumption of ground juniper containing supplements. Results also provide novel information regarding the effects of plant secondary compound consumption throughout pregnancy on ewe and progeny performance and health.

Mitochondrial glycerol 3-phosphate facilitates bumblebee pre-flight thermogenesis.

Bumblebees (Bombus terrestris) fly at low ambient temperatures where other insects cannot, and to do so they must pre-warm their flight muscles. While some have proposed mechanisms, none fully explain how pre-flight thermogenesis occurs. Here, we present a novel hypothesis based on the less studied mitochondrial glycerol 3-phosphate dehydrogenase pathway (mGPDH). Using calorimetry, and high resolution respirometry coupled with fluorimetry, we report substrate oxidation by mGPDH in permeabilised flight muscles operates, in vitro, at a high flux, even in the absence of ADP. This may be facilitated by an endogenous, mGPDH-mediated uncoupling of mitochondria. This uncoupling increases ETS activity, which results in increased heat release. Furthermore, passive regulation of this mechanism is achieved via dampened temperature sensitivity of mGPDH relative to other respiratory pathways, and subsequent consumption of its substrate, glycerol 3-phosphate (G3P), at low temperatures. Mitochondrial GPDH may therefore facilitate pre-flight thermogenesis through poor mitochondrial coupling. We calculate this can occur at a sufficient rate to warm flight muscles until shivering commences, and until flight muscle function is adequate for bumblebees to fly in the cold.

A powerful test of independent assortment that determines genome-wide significance quickly and accurately.

In the analysis of DNA sequences on related individuals, most methods strive to incorporate as much information as possible, with little or no attention paid to the issue of statistical significance. For example, a modern workstation can easily handle the computations needed to perform a large-scale genome-wide inheritance-by-descent (IBD) scan, but accurate assessment of the significance of that scan is often hindered by inaccurate approximations and computationally intensive simulation. To address these issues, we developed gLOD-a test of co-segregation that, for large samples, models chromosome-specific IBD statistics as a collection of stationary Gaussian processes. With this simple model, the parametric bootstrap yields an accurate and rapid assessment of significance-the genome-wide corrected P-value. Furthermore, we show that (i) under the null hypothesis, the limiting distribution of the gLOD is the standard Gumbel distribution; (ii) our parametric bootstrap simulator is approximately 40 000 times faster than gene-dropping methods, and it is more powerful than methods that approximate the adjusted P-value; and, (iii) the gLOD has the same statistical power as the widely used maximum Kong and Cox LOD. Thus, our approach gives researchers the ability to determine quickly and accurately the significance of most large-scale IBD scans, which may contain multiple traits, thousands of families and tens of thousands of DNA sequences.

Effects of Juniperus species and stage of maturity on nutritional, in vitro digestibility, and plant secondary compound characteristics.

Rising feed costs and recurring feed shortages necessitate the investigation into alternative and underutilized feed resources. Nutritional characteristics of species are either unknown or limited to leaves and ground material from small stems. Therefore, the objective was to quantify nutritional characteristics, 48-h true IVDMD (tIVDMD), microbial gas production, and secondary compound characteristics of entire woody plant material of 4 species-, , , and -at immature and mature stages of growth. Immature plants had greater CP concentrations and lower NDF concentrations ( < 0.001) than mature plants regardless of species. Mature plants also had greater ( < 0.001) concentrations of ADF compared with immature plants with the exception of . In general, immature , , and had greater ( < 0.02) tIVDMD and total 48-h and asymptotic gas production than mature plants. Immature and plants were more digested (tIVDMD; < 0.001) than immature and , but tIVDMD did not differ in mature plant material across species. Condensed tannins (CT) were greater ( < 0.001) in immature and than mature plants; differences in CT concentrations among immature species were also detected ( < 0.04). Volatile oil yields were similar across maturity and species with 1 exception: immature yielded more ( < 0.02) volatile oil than mature material. Volatile oil composition across species varied and contained a range of 65 to 70 terpene compounds. The dominant terpenes across species were generally greater ( < 0.05) in immature vs. mature plant material with the exception of . Labdane acids were negligible in , , and and greater in ( < 0.001). Ground material from mature juniper species, although inferior in nutritional quality compared with immature plants, is comparable to traditional low-quality roughage ingredients. Given that has been successfully fed in lamb feedlot diets, the similarities of , and suggest that all three species have potential to be effective roughage ingredients.