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1280 Liver proteomic analysis of cows exposed to heat stress or cooling conditions during the dry period.
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Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.
Copyright © 2015. Published by Elsevier B.V.
The liver is the main metabolic organ coordinating the adaptations that take place during the peripartal period of dairy cows. A successful transition into lactation, rather than management practices alone, depends on environmental factors such as temperature, season of parturition, and photoperiod. Therefore, we analyzed the effect of calving season on the hepatic transcriptome of dairy cows during the transition period. A total of 12 Holstein dairy cows were assigned into 2 groups based on calving season (6 cows March-April, spring; 6 cows June-July, summer, SU). The RNA was extracted from liver samples obtained at -30, 3, and 35 DIM via percutaneous biopsy and hybridized to the Agilent 44K Bovine (V2) Gene Expression Microarray (Agilent Technologies Inc., Santa Clara, CA). A quantitative PCR on 22 target genes was performed to verify and expand the analyses. A total of 4,307 differentially expressed genes were detected (false discovery rate ≤0.05) in SU compared with spring. Furthermore, 73 unique differentially expressed genes were detected in SU compared with spring cows after applying a fold-change threshold ≥3 and ≤-3. For Kyoto Encyclopedia of Genes and Genomes pathways analysis of differentially expressed genes, we used the dynamic impact approach. Ingenuity Pathway Analysis software was used to analyze upstream transcription regulators and perform gene network analysis. Among metabolic pathways, energy metabolism from lipids, carbohydrates, and amino acids was strongly affected by calving in SU, with a reduced level of fatty acid synthesis, oxidation, re-esterification, and synthesis of lipoproteins, leading to hepatic lipidosis. Glycan-synthesis was downregulated in SU cows probably as a mechanism to counteract the progression of this lipidosis. In contrast, calving in the SU resulted in upregulation of gluconeogenesis but also greater use of glucose as an energy source. Among nonmetabolic pathways, the heat-shock response was obviously activated in SU cows but was also associated with inflammatory and intracellular stress response. Furthermore, data support a recent finding that cows experience endoplasmic reticulum stress around parturition. Transcription regulator analysis revealed how metabolic changes are related to important regulatory mechanisms, including epigenetic modification. The holistic analyses of the liver transcriptome response to calving in the summer at high environmental temperatures underscore how transition cows should be carefully managed during this period, as they experience alterations in liver energy metabolism and inflammatory state increasing susceptibility to health disorders in early postpartum.
Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Carbon-based supercapacitors can provide high electrical power, but they do not have sufficient energy density to directly
compete with batteries. We found that a nitrogen-doped ordered mesoporous few-layer carbon has a capacitance of 855 farads
per gram in aqueous electrolytes and can be bipolarly charged or discharged at a fast, carbon-like speed. The improvement
mostly stems from robust redox reactions at nitrogen-associated defects that transform inert graphene-like layered carbon
into an electrochemically active substance without affecting its electric conductivity. These bipolar aqueous-electrolyte
electrochemical cells offer power densities and lifetimes similar to those of carbon-based supercapacitors and can store a
specific energy of 41 watt-hours per kilogram (19.5 watt-hours per liter).
Significance
Mammalian complex I, the largest and most complicated enzyme of the mitochondrial respiratory chain, is an L-shaped assembly of 44 proteins with one arm in the mitochondrial matrix and the orthogonal arm buried in the inner membrane. It is put together from preassembled modules. This investigation concerns the little studied process of the assembly of the membrane arm module from proteins emanating from both nuclear and mitochondrial genomes. We have identified two membrane protein assembly factors C3orf1 and TMEM126B, not found in the mature complex, that help this process by putting together two membrane arm subcomplexes. Defects in the assembly of complex I are increasingly being associated with human pathologies.
Environmental-induced hyperthermia compromises efficient animal production and jeopardizes animal welfare. Reduced productive output during heat stress was traditionally thought to result from decreased nutrient intake. Our observations challenge this dogma and indicate that heat-stressed animals employ novel homeorhetic strategies to direct metabolic and fuel selection priorities independent of nutrient intake or energy balance. Alterations in systemic physiology support a shift in carbohydrate metabolism, evident through changes such as basal and stimulated circulating insulin levels. Hepatocyte and myocyte metabolism also show clear differences in glucose production and use during heat stress. Perhaps most intriguing, given the energetic shortfall of the heat-stressed animal, is the apparent lack of fat mobilization from adipose tissue coupled with a reduced responsiveness to lipolytic stimuli. Thus, the heat stress response markedly alters postabsorptive carbohydrate, lipid, and protein metabolism ind...
Our objective was to provide a review of factors influencing heat stress in lactating dairy cows and how it affects milk production. In warmer parts of the world, during summer months in the United States, and in other temperate regions, reduced milk production resulting from heat stress counteracts tremendous genetic progress achieved in increasing milk production. Genetic progress in milk production is closely related to increased feed intake. High feed intake results in raised metabolic heat increment. High metabolic heat increment requires effective thermoregulatory mechanisms to maintain body temperature in a thermoneutral zone and in physiological homeostasis. Cows can succumb to hyperthermia if they fail to maintain thermoneutrality. Accurate measurement of when cows enter heat stress is complicated because the responses to heat stress affect not only the energy balance, but also water, sodium, potassium and chlorine metabolism. Water, sodium, potassium and chlorine are important constituents of sweat, and sweating is a major, if not the most important, thermoregulatory mechanism used to dissipate excess body heat. Due to high metabolic heat increment, and especially in the warmer months, high-producing dairy cows may enter heat stress much earlier than their lower-producing counterparts and than currently thought, or extra heat has been accommodated by physiological adaptations. Should this be the case, then strategies to reduce heat stress must be developed to enable cows to express their full genetic potential. The thermoneutral zone, heat production and heat gain, heat dissipation mechanisms, and how the lactating cow responds to heat stress are discussed.
Environmentally induced periods of heat stress decrease productivity with devastating economic consequences to global animal agriculture. Heat stress can be defined as a physiological condition when the core body temperature of a given species exceeds its range specified for normal activity, which results from a total heat load (internal production and environment) exceeding the capacity for heat dissipation and this prompts physiological and behavioral responses to reduce the strain. The ability of ruminants to regulate body temperature is species- and breed-dependent. Dairy breeds are typically more sensitive to heat stress than meat breeds, and higher-producing animals are more susceptible to heat stress because they generate more metabolic heat. During heat stress, ruminants, like other homeothermic animals, increase avenues of heat loss and reduce heat production in an attempt to maintain euthermia. The immediate responses to heat load are increased respiration rates, decreased feed intake and increased water intake. Acclimatization is a process by which animals adapt to environmental conditions and engage behavioral, hormonal and metabolic changes that are characteristics of either acclimatory homeostasis or homeorhetic mechanisms used by the animals to survive in a new 'physiological state'. For example, alterations in the hormonal profile are mainly characterized by a decline and increase in anabolic and catabolic hormones, respectively. The response to heat load and the heat-induced change in homeorhetic modifiers alters post-absorptive energy, lipid and protein metabolism, impairs liver function, causes oxidative stress, jeopardizes the immune response and decreases reproductive performance. These physiological modifications alter nutrient partitioning and may prevent heat-stressed lactating cows from recruiting glucose-sparing mechanisms (despite the reduced nutrient intake). This might explain, in large part, why decreased feed intake only accounts for a minor portion of the reduced milk yield from environmentally induced hyperthermic cows. How these metabolic changes are initiated and regulated is not known. It also remains unclear how these changes differ between short-term v. long-term heat acclimation to impact animal productivity and well-being. A better understanding of the adaptations enlisted by ruminants during heat stress is necessary to enhance the likelihood of developing strategies to simultaneously improve heat tolerance and increase productivity.
Heat stress during the dry period negatively affects hepatic metabolism and cellular immune function during the transition period, and milk production in the subsequent lactation. However, the cellular mechanisms involved in the depressed mammary gland function remain unknown. The objective of the present study was to determine the effect of heat stress during the dry period on various indices of mammary gland development of multiparous cows. Cows were dried off approximately 46 d before expected calving and randomly assigned to 2 treatments, heat stress (HT, n=15) or cooling (CL, n=14), based on mature equivalent milk production. Cows in the CL treatment were provided with sprinklers and fans that came on when ambient temperatures reached 21.1°C, whereas HT cows were housed in the same barn without fans and sprinklers. After parturition, all cows were housed in a freestall barn with cooling. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rates recorded at 1500 h on a Monday-Wednesday-Friday schedule from dry off to calving. Milk yield and composition were recorded daily up to 280 d in milk. Daily dry matter intake was measured from dry off to 42 d relative to calving. Mammary biopsies were collected at dry off, -20, 2, and 20 d relative to calving from a subset of cows (HT, n=7; CL, n=7). Labeling with Ki67 antigen and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling were used to evaluate mammary cell proliferation and apoptosis, respectively. The average temperature-humidity index during the dry period was 76.6 and not different between treatments. Heat-stressed cows had higher rectal temperatures in the morning (38.8 vs. 38.6°C) and afternoon (39.4 vs. 39.0°C), greater respiration rates (78.4 vs. 45.6 breath/min), and decreased dry matter intake (8.9 vs. 10.6 kg/d) when dry compared with CL cows. Relative to HT cows, CL cows had greater milk production (28.9 vs. 33.9 kg/d), lower milk protein concentration (3.01 vs. 2.87%), and tended to have lower somatic cell score (3.35 vs. 2.94) through 280 d in milk. Heat stress during the dry period decreased mammary cell proliferation rate (1.0 vs. 3.3%) at -20 d relative to calving compared with CL cows. Mammary cell apoptosis was not affected by prepartum heat stress. We conclude that heat stress during the dry period compromises mammary gland development before parturition, which decreases milk yield in the next lactation.
In mammals, complex I (NADH-ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, makes complex I a sensible pace-maker of mitochondrial respiration. The work reviewed here shows that the cAMP/PKA pathway regulates the biogenesis, assembly and catalytic activity of complex I and mitochondrial oxygen superoxide production. The structural, functional and regulatory complexity of complex I, renders it particularly vulnerable to genetic and sporadic pathological factors. Complex I dysfunction has, indeed, been found, to be associated with several human diseases. Knowledge of the pathogenetic mechanisms of these diseases can help to develop new therapeutic strategies.
Growing evidence indicates that chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is integral in the development of cardiovascular diseases (CVD). These ROS can be released from nicotinamide adenine dinucleotide (phosphate) oxidase, xanthine oxidase, lipoxygenase, mitochondria, or the uncoupling of nitric oxide synthase in vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis: from the initiation of fatty streak development through lesion progress to ultimate plaque rupture. Various animal models of oxidative stress support the notion that ROS have a causal role in atherosclerosis and other cardiovascular diseases. Human investigations also support the oxidative stress hypothesis of atherosclerosis. Oxidative stress is the unifying mechanism for many CVD risk factors, which additionally supports its central role in CVD. Despite the demonstrated role of antioxidants in cellular and animal studies, the ineffectiveness of antioxidants in reducing cardiovascular death and morbidity in clinical trials has led many investigators to question the importance of oxidative stress in human atherosclerosis. Others have argued that the prime factor for the mixed outcomes from using antioxidants to prevent CVD may be the lack of specific and sensitive biomarkers by which to assess the oxidative stress phenotypes underlying CVD. A better understanding of the complexity of cellular redox reactions, development of a new class of antioxidants targeted to specific subcellular locales, and the phenotype-genotype linkage analysis for oxidative stress will likely be avenues for future research in this area as we move toward the broader use of pharmacological and regenerative therapies in the treatment and prevention of CVD.
AMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. Here we demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and -2). Ser372 phosphorylation of SREBP-1c by AMPK is necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin-resistant LDL receptor-deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c- and -2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis.
Study objectives were to evaluate hepatic gluconeogenic enzyme gene expression in recombinant bovine somatotropin (rbST)-treated lactating dairy cattle during heat stress (HS) or in thermal-neutral, pair-fed (PF) animals. Twenty-two multiparous (99 d in milk, 656 kg of BW) Holstein cows were subjected to 3 consecutive experimental periods (7 d each): (1) thermal neutral, (2) HS or PF, and (3) HS or PF with rbST (Posilac, administered on d 1 of period 3). Liver biopsies were obtained on the final day of each period. Heat stress conditions progressively decreased dry matter intake for the first 5 to 6 d during period 2 before stabilizing (a decrease of 6.15 kg; 30%) on d 6 and 7, and feed intake remained stable and not different from period 2 during period 3. Cytosolic phosphoenolpyruvate carboxykinase mRNA abundance increased during PF, but was unaffected by HS or bST. Pyruvate carboxylase gene expression increased during HS and PF, and administering bST decreased pyruvate carboxylase mRNA abundance during both HS and PF. Insulin-like growth factor-I gene expression increased following bST administration during HS and PF, confirming hepatic bST responsiveness. Exposure to HS leads to a change in hepatic gluconeogenic enzyme profile that appears to be dependent on plane of nutrition.
Heat stress has an enormous economic impact on the global dairy industry, but the mechanisms by which hyperthermia negatively affect systemic physiology and milk synthesis are not clear. Study objectives were to evaluate production parameters and metabolic variables in lactating dairy cows during short-term heat stress or pair-fed conditions coupled with bST administration. Twenty-two multiparous Holstein cows were subjected to 3 experimental periods: 1) thermoneutral conditions with ad libitum intake for 7 d (P1); 2) heat stress (HS) with ad libitum intake (n=10) or pair-fed (PF) in thermoneutral conditions (n=12) for 7 d (P2), and 3) 7 d of HS or PF in conditions as described in P2 with recombinant bovine somatotropin administered on d 1 (P3). All cows received an intravenous glucose tolerance test (GTT) on d 5 of each period. Heat stress conditions were cyclical and temperatures ranged from 29.4 to 38.9 degrees C. Rectal temperatures and respiration rates increased during heat stress (38.6-40.4 degrees C and 44-89 breaths/min, respectively). Heat stress reduced dry matter intake by 30% and by design PF cows had similar intake reductions (28%). During heat stress and pair-feeding, milk yield decreased by 27.6% (9.6kg) and 13.9% (4.8kg), respectively, indicating that reduced feed intake accounted for only 50% of the decreased milk production. Milk yield increased with recombinant bovine somatotropin in both HS (9.7%) and PF (16.1%) cows. Cows in both groups were in positive energy balance (3.95 Mcal/d) during P1 but entered negative energy balance during P2 and P3 (-5.65 Mcal/d). Heat stress and pair-feeding treatments decreased (9.3%) basal glucose concentrations. Heat stress conditions had no effect on basal NEFA levels during P2; however, PF cows (despite a similar calculated energy balance) had a 2-fold increase in basal NEFA concentrations. Both groups had increased plasma urea nitrogen levels during P2 and P3 compared with P1. Basal insulin levels increased (37%) during P2 and P3 in HS cows but did not differ between periods in PF cows. During P2 and compared with P1, PF cows had a decreased rate of glucose disposal, whereas HS cows had a similar disposal rate following the GTT. During P2 and compared with P1, PF cows had a reduced insulin response whereas HS cows had a similar insulin response to the GTT. In summary, reduced nutrient intake accounted for only 50% of heat stress-induced decreases in milk yield, and feed intake-independent shifts in postabsorptive glucose and lipid homeostasis may contribute to the additional reduction in milk yield.
This MiniReview summarizes the available data on the metabolic role of PEPCK and emphasizes the role of both isoforms of the enzyme.
Heat stress is detrimental to dairy production and affects numerous variables including feed intake and milk production. It is unclear, however, whether decreased milk yield is primarily due to the associated reduction in feed intake or the cumulative effects of heat stress on feed intake, metabolism, and physiology of dairy cattle. To distinguish between direct (not mediated by feed intake) and indirect (mediated by feed intake) effects of heat stress on physiological and metabolic indices, Holstein cows (n = 6) housed in thermal neutral conditions were pair-fed (PF) to match the nutrient intake of heat-stressed cows (HS; n = 6). All cows were subjected to 2 experimental periods: 1) thermal neutral and ad libitum intake for 9 d (P1) and 2) HS or PF for 9 d (P2). Heat-stress conditions were cyclical with daily temperatures ranging from 29.7 to 39.2 degrees C. During P1 and P2 all cows received i.v. challenges of epinephrine (d 6 of each period), and growth hormone releasing factor (GRF; d 7 of each period), and had circulating somatotropin (ST) profiles characterized (every 15 min for 6 h on d 8 of each period). During P2, HS cows were hyperthermic for the entire day and peak differences in rectal temperatures and respiration rates occurred in the afternoon (38.7 to 40.2 degrees C and 46 to 82 breaths/min, respectively). Heat stress decreased dry matter intake by greater than 35% and, by design, PF cows had similar reduced intakes. Heat stress and PF decreased milk yield, although the pattern and magnitude (40 and 21%, respectively) differed between treatments. The reduction in dry matter intake caused by HS accounted for only approximately 35% of the decrease in milk production. Both HS and PF cows entered into negative energy balance, but only PF cows had increased (approximately 120%) basal nonesterified fatty acid (NEFA) concentrations. Both PF and HS cows had decreased (7%) plasma glucose levels. The NEFA response to epinephrine did not differ between treatments but was increased (greater than 50%) in all cows during P2. During P2, HS (but not PF) cows had a modest reduction (16%) in plasma insulin-like growth factor-I. Neither treatment nor period had an effect on the ST response to GRF and there was little or no treatment effect on mean ST levels or pulsatility characteristics, but both HS and PF cows had reduced mean ST concentrations during P2. In summary, reduced nutrient intake accounted for just 35% of the HS-induced decrease in milk yield, and modest changes in the somatotropic axis may have contributed to a portion of the remainder. Differences in basal NEFA between PF and HS cows suggest a shift in postabsorptive metabolism and nutrient partitioning that may explain the additional reduction in milk yield in cows experiencing a thermal load.
Mitochondria are essential for cellular bioenergetics by way of energy production in the form of ATP through the process of oxidative phosphorylation. This crucial task is executed by five multi-protein complexes of which mitochondrial NADH:ubiquinone oxidoreductase or complex I is the largest and most complicated one. During recent years, mutations in nuclear genes encoding structural subunits of complex I have been identified as a cause of devastating neurodegenerative disorders with onset in early childhood. Here, we present a comprehensive overview of clinical, biochemical and cell physiological information of 15 children with isolated, nuclear-encoded complex I deficiency, which was generated in a joint effort of clinical and fundamental research. Our findings point to a rather homogeneous clinical picture in these children and drastically illustrate the severity of the disease. In extensive live cell studies with patient-derived skin fibroblasts we uncovered important cell physiological aspects of complex I deficiency, which point to a central regulatory role of cellular reactive oxygen species production and altered mitochondrial membrane potential in the pathogenesis of the disorder. Moreover, we critically discuss possible interconnections between clinical signs and cellular pathology. Finally, our results indicate apparent differences to drug therapy on the cellular level, depending on the severity of the catalytic defect and identify modulators of cellular Ca(2+) homeostasis as new candidates in the therapy of complex I deficiency.
The liver of dairy cows is involved in signaling the current hepatic metabolic state to the brain via metabolites and nerval afferents to control and adjust feed intake. Feed deprivation may result in mobilization of body reserves favoring hepatic steatosis. While the overall metabolic changes are well characterized, specific regulatory mechanisms are not readily understood. To identify molecular events associated with metabolic adaptation and the control of energy homeostasis, liver specimens from six ad libitum-fed and six feed-deprived cows were analyzed for selected metabolites, for the activation of AMP kinase, and for regulatory/regulated proteins using two-dimensional gel electrophoresis and MALDI-TOF-MS. Feed deprivation increased total liver fat and the calcium content, as well as augmented AMPK phosphorylation, while it decreased the contents of protein, glucose, glycogen, and cholesterol when expressed as a percentage of dry matter. Among 34 differentially expressed proteins identified, we found downregulation of proteins associated with fatty acid oxidation, glycolysis, electron transfer, protein degradation, and antigen processing, as well as cytoskeletal rearrangement. Proteins upregulated after feed deprivation included enzymes of the urea cycle, fatty acid or cholesterol transport proteins, an inhibitor of glycolysis, and previously unknown changes in calcium signaling network. Direct correlation was found between expression of glycolytic enzymes and glucose/glycogen content, whereas inverse correlation exists between expression of beta-oxidative enzymes and total liver fat content. In conclusion, the regulatory response of identified proteins may help to explain development and consequences of hepatic lipidosis but also offers novel candidates potentially involved in signaling for maintaining energy homeostasis.
A major mechanism in the cellular defense against oxidative or electrophilic stress is activation of the Nrf2-antioxidant
response element signaling pathway, which controls the expression of genes whose protein products are involved in the detoxication
and elimination of reactive oxidants and electrophilic agents through conjugative reactions and by enhancing cellular antioxidant
capacity. At the molecular level, however, the regulatory mechanisms involved in mediating Nrf2 activation are not fully understood.
It is well established that Nrf2 activity is controlled, in part, by the cytosolic protein Keap1, but the nature of this pathway
and the mechanisms by which Keap1 acts to repress Nrf2 activity remain to be fully characterized and are the topics of discussion
in this minireview. In addition, a possible role of the Nrf2-antioxidant response element transcriptional pathway in neuroprotection
will also be discussed.
[Alpha-13C]- and [alpha,beta-13C]valine were administered sequentially to a patient with methylmalonicacidemia to clarify the metabolic pathway of valine from methylmalonic acid semialdehyde to methylmalonyl-CoA. Methylmalonic acid was isolated from multiple urine samples, purified, and analyzed by 13C nuclear magnetic resonance spectroscopy. Contrary to the widely accepted view, the results show unequivocally that methylmalonic acid semialdehyde is decarboxylated to propionate before conversion to methylmalonyl CoA.
The genes coding for three proteins of the plasma lipid transport system--apolipoproteins A1 (APOA1), C3 (APOC3), and A4 (APOA4)--are closely linked and tandemly organized on the long arm of human chromosome 11. In this study the human APOA4 gene has been isolated and characterized. In contrast to APOA1 and APOC3 genes, which contain three introns, the APOA4 gene contains only two. An intron interrupting the 5' noncoding region of the APOA1 and APOC3 mRNAs is absent from the corresponding position of the APOA4 mRNA. However, similar to APOA1 and APOC3 genes, the introns of the APOA4 gene separate nucleotide sequences coding for the signal peptide and the amphipathic domains in APOA4. These results suggest that the APOA1, APOC3, and APOA4 genes were derived from a common evolutionary ancestor and indicate that during evolution the APOA4 gene lost one of its ancestral introns. Two restriction endonuclease sites, an Xba I located in the second intron of the APOA4 gene and a different Xba I located 9 kilobases 3' to the APOA4 gene, are polymorphic in Mediterranean and Northern European populations. Haplotype analysis indicated that even though these polymorphic sites are located within 9 kilobases they do not display significant nonrandom association. Finally, restriction mapping analysis of DNA from a patient with combined APOA1-APOC3 deficiency and premature coronary artery disease indicated that this patient has a structurally normal APOA4 gene.
Conceptus energy and nitrogen demands in late pregnancy are mostly met by placental uptake of maternal glucose and amino acids. The resulting 30 to 50% increase in maternal requirements for these nutrients is met partly by increased voluntary intake and partly by an array of maternal metabolic adaptations. The latter include increased hepatic gluconeogenesis from endogenous substrates, decreased peripheral tissue glucose utilization, increased fatty acid mobilization from adipose tissue, and, possibly, increased amino acid mobilization from muscle. Within 4 d of parturition, mammary demands for glucose, amino acids, and fatty acids are several-fold those of the pregnant uterus before term. Even unusual postparturient increases in voluntary intake cannot satisfy this increased nutrient demand. Therefore, rates of hepatic gluconeogenesis and adipose fat mobilization are greatly accelerated. Concomitant changes in amino acid metabolism include increased hepatic protein synthesis and, possibly, decreased amino acid catabolism, and increased peripheral mobilization of amino acids. Insulin resistance in adipose tissue and muscle, developed during late pregnancy, continues postpartum; adipose lipolytic responsiveness and sensitivity to adrenergic agents are increased postpartum beyond their levels during late pregnancy. Before parturition, these homeorhetic adjustments may be coordinated with lactogenesis by increased secretion of estradiol and prolactin. Their amplification and reinforcement at and soon after parturition may be regulated mostly by somatotropin.
Heat stress during late gestation in cattle negatively impacts the performance of the dam and its calf. This brief exposure to an adverse environment before parturition impacts the physiological responses, tissue development, metabolism and immune function of the dam and her offspring, thereby limiting their productivity. During the dry period of a dairy cow, heat stress blunts mammary involution by attenuating mammary apoptosis and autophagic activity, and reduces subsequent mammary cell proliferation, leading to impaired milk production in the next lactation. Dairy cows in early lactation that experience prepartum heat stress display reduced adipose tissue mobilization and lower degree of insulin resistance in peripheral tissues. Similar to mammary gland development, placental function is impaired by heat stress as evidenced by reduced secretion of placental hormones (e.g., estrone sulfate) in late gestation cows, which partly explains the reduced fetal growth rate and lighter birth weight of the calves. Compared with dairy calves born to dams that are exposed to evaporative cooling during summer, calves born to non-cooled dry cows maintain lower body weight until 1 yr of age, but display a stronger ability to absorb glucose during metabolic challenges postnatally. Immunity of the calves, both passive and cell-mediated immune function, is also impaired by prenatal heat stress, resulting in increased susceptibility of the calves to diseases in their postnatal life. In fact, dairy heifers born to heat-stressed dry cows without evaporative cooling have a greater chance leaving the herd before puberty compared with heifers born to dry cows provided with evaporative cooling (12.2 vs. 22.7%). Dairy heifers born to late gestation heat-stressed dry cows have lower milk yield at maturity during their first and second lactations. Emerging evidence suggest that late gestation heat stress alters the mammary gland microstructure of the heifers during the first lactation, and exerts epigenetic alterations that might explain, in part their impaired productivity.
Significance:
This work shows that seasonal heat stress increases plasma concentrations of the oxidative stress marker malondialdehyde and cortisol in transition dairy cows. As many proteins expressed in the adipose tissue are involved in metabolic responses to stress, we investigated the effects of heat stress on the proteome of adipose tissue from late-pregnant cows during summer or winter seasons. We demonstrated that heat stress enriches several stress-related pathways, such as the Nrf2-mediated oxidative stress response and the acute-phase response in adipose tissues. Thus, environmental heat stress has a unique effect on adipose tissue in late-pregnant cows, as part of the regulatory adaptations to chronic heat load during the summer season. In addition, this study presents the widest available dataset of adipose tissue proteome in dairy cows, and revealed several novel biomarkers of heat stress in adipose tissue of dairy cows, the use of which awaits further validation.
Significance:
High yielding dairy cows suffer from infertility that leads to major economic losses worldwide. In Israel, about 30% of dairy cows fail to conceive following ≥4 inseminations. The etiology of this low fertility is multifactorial and remains a serious challenge. Follicular fluid proteome can provide indication to follicular quality, yet the proteome of pre-ovulatory follicles has not been described in cows. This work examined the differential abundance of proteins in less fertile dairy cows compared to controls, and found 8 relevant novel proteins that could influence follicular function. The role of these proteins in the etiology of less fertile cows should be further examined.
Adipose tissue is a central regulator of metabolism in dairy cows. However, little is known about the association between various proteins in adipose tissue and the metabolic status of peripartum cows. Therefore, the objectives were to: 1) examine total protein expression in adipose tissue of dairy cows, and 2) identify biomarkers in adipose that are linked to insulin resistance and to cows' metabolic status. Adipose tissue biopsies were obtained from 8 multiparous cows at -17 and +4 d relative to parturition. Proteins were analyzed by intensity-based, label-free, quantitative shotgun proteomics (nanoLC-MS/MS). Cows were divided into groups with insulin-resistant (IR) and insulin-sensitive (IS) adipose according to protein kinase B phosphorylation following insulin stimulation. Cows with IR adipose lost more body weight postpartum compared to IS cows. Differential expression of 143 out of 586 proteins was detected in prepartum vs. postpartum adipose. Comparing IR to IS adipose revealed differential expression of 18.9% of the proteins; those related to lipolysis (hormone-sensitive lipase, perilipin, monoglycerol lipase) were increased in IR adipose. In conclusion, we found novel biomarkers related to IR in adipose and to metabolic status that could be used to characterize high-yielding dairy cows that are better adapted to peripartum metabolic stress.
Complex I (CI) deficiency is the most common respiratory chain defect representing more than 30% of mitochondrial diseases. CI is a L-shaped multi-subunit complex with a peripheral arm protruding into the mitochondrial matrix and a membrane arm. CI sequentially assembled into main assembly intermediates: the P (pumping), Q (Quinone) and N (NADH dehydrogenase) modules. In this study, we analysed 11 fibroblast cell lines derived from patients with inherited CI deficiency resulting from mutations in the nuclear or mitochondrial DNA and impacting these different modules. In patient cells carrying a mutation located in the matrix arm of CI, blue native-polyacrylamide gel electrophoresis (BN-PAGE) revealed a significant reduction of fully assembled CI enzyme and an accumulation of intermediates of the N module. In these cell lines with an assembly defect, NADH dehydrogenase activity was partly functional, even though CI was not fully assembled. We further demonstrated that this functional N module was responsible for ROS production through the reduced flavin mononucleotide. Due to the assembly defect, the FMN site was not re-oxidized leading to a significant oxidative stress in cell lines with an assembly defect. These findings not only highlight the relationship between CI assembly and oxidative stress, but also show the suitability of BN-PAGE analysis in evaluating the consequences of CI dysfunction. Moreover, these data suggest that the use of antioxidants may be particularly relevant for patients displaying a CI assembly defect.
Copyright © 2015. Published by Elsevier Ltd.
Presented is a data set for benchmarking MS1 based label free quantitative proteomics using a quadrupole orbitrap mass spectrometer. Escherichia coli digest was spiked into a HeLa digest in four different concentrations, simulating protein expression differences in a background of an unchanged complex proteome. The data set provides a unique opportunity to evaluate the proteomic platform (instrumentation and software) in its ability to perform MS1 intensity based label free quantification. We show that the presented combination of informatics and instrumentation produces high precision and quantification accuracy. The data was also used to compare different quantitative protein inference methods such as iBAQ and Hi-N. The data can also be used as a resource for development and optimization of proteomics informatics tools, thus the raw data have been deposited to ProteomeXchange with identifier PXD001385.
Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved 'core' subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 'supernumerary' subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.
Mammalian Complex I is composed of fourteen highly conserved core subunits and additional thirty subunits acquired in the course of evolution. At present, the function of the majority of these supernumerary subunits is poorly understood. In this work, we have studied NDUFA3, NDUFA5 and NDUFA12 supernumerary subunits to gain insight into their role in CI activity and biogenesis. Using human cell lines in which the expression of these subunits was knocked down with miRNAs, we showed that they are necessary for the formation of a functional holoenzyme. Analysis of the assembly intermediates in mitochondria depleted for these subunits further suggested that they are required for assembly and/or stability of the electron transferring Q module in the peripheral arm of the CI.
Previous studies demonstrated that apolipoprotein A-IV (apoA-IV) promotes apoB lipoprotein-mediated triglyceride (TG) secretion in transfected enterocytes and hepatoma cells; however, evidence for a role in lipid transport in vivo is lacking. Using mouse models, we explored the role of apoA-IV in hepatic very-low-density lipoprotein-mediated lipid efflux under conditions that promote hepatic steatosis.
Hepatic steatosis, induced by either high-fat diet or enhanced de novo lipogenesis caused by transgenic overexpression of SREBP-1a (SREBP-1a(Tg)), was associated with up to a 43-fold induction of hepatic apoA-IV mRNA and protein levels. In both models, a positive linear correlation between hepatic TG content and apoA-IV mRNA abundance was observed (r(2)=0.8965). To examine whether induction of apoA-IV affected hepatic TG secretion, SREBP-1a(Tg) mice were crossed with Apoa4 knockout mice. With Triton blockade of peripheral lipolysis, SREBP-1a(Tg)/Apoa4 knockout mice demonstrated a 24% reduction in hepatic TG secretion rate, relative to SREBP-1a(Tg) controls, but no change in apoB production. Negative stain electron microscopy revealed a 33% decrease in the abundance of secreted very-low-density lipoprotein particles with diameters ≥120 nm. Conversely, mice infected with a recombinant human apoA-IV adenovirus demonstrated a 52% increase in the hepatic TG secretion rate, relative to controls, a 38% reduction in liver TG content, and a 43% increase in large diameter (≥120 nm) very-low-density lipoprotein particles, with no change in apoB secretion.
Hepatic steatosis in mice induces hepatic apoA-IV expression, which in turn promotes lipoprotein particle expansion and reduces hepatic lipid burden without increasing the number of secreted atherogenic apoB-containing lipoprotein particles.
Heat stress is recognized as one of the main factors adversely affecting yield, reproduction and health of dairy ruminants. The peripartum period is the most critical phase for dairy cows. The interaction between heat stress and physiological stress imposed by the peripartum, might worsen the metabolic status of transition dairy cows. Therefore, the objective of the present study was to investigate the effect of hot season on metabolic status and on hepatic apolipoprotein B100 (ApoB100) expression during the peripartum period in Holstein dairy cows. Twenty-four multiparous Holstein cows were utilized in the study. Twelve cows gave birth in spring (SP: 28 March to 30 April). The remaining 12 cows gave birth in summer (SU: 15 June to 2 July). The two groups were balanced for parity and body condition score (BCS) and monitored from −30 to 35d relative to calving. Values of air temperature (°C) and relative humidity (%) were recorded at 1h intervals and were utilized to calculate the temperature–humidity index (THI). Milk production and BCS were recorded weekly. Blood samples were taken at 0800h from the jugular vein −30, −18, and −4d prepartum and 3, 17, and 35d postpartum. Plasma was analyzed for glucose, urea, cholesterol, β-OH butyrate (BHBA), non-esterified fatty acids (NEFA) and ApoB100. Liver biopsies were performed on d −30, 3, and 35 relative to day of calving, and tissue samples were used for histopathological examination, and for ApoB100 mRNA and protein determination. Milk yield in SU cows was lower than in their SP counterparts. During summer, transition cows showed lower glucose and cholesterol, higher NEFA and BHBA, had lower liver ApoB100 gene and protein expression and higher lipid accumulation in liver compared with transition cows during spring. The current study provides the first evidence about the negative effects of hot season on ApoB100 gene and protein expression in transition dairy cows. The down-regulation of ApoB100 expression together with higher circulating NEFA and altered lipid metabolism are likely to be responsible for the higher risk of liver lipidosis in transition SU cows. These metabolic conditions make transition cows more susceptible to metabolic diseases during hot season.
The transition period, from 3 wk before to 3 wk after parturition, is critically important to health, production, and profitability of dairy cows. Most health disorders occur during this time. Compared with other stages of the lactation cycle, relatively little is known about fundamental biological processes during the transition period. The regulation and coordination of lipid metabolism among adipose tissue, liver, gut, and mammary gland are key components of the adaptations to lactation. Lipid accumulation in liver may contribute to health disorders and decreased milk production. Knowledge of key control points in hepatic metabolism of long-chain fatty acids is lacking, as is an understanding of the metabolic effects of hormones, growth factors, and cytokines that mediate stress. Recent evidence indicates that supplemental fats or restricted intakes before parturition can induce a coordinated set of metabolic changes in metabolism of long-chain fatty acids, including peroxisomal β-oxidation, perhaps mediated by peroxisome proliferator-activated receptors. Estimates of the mixture of fuels constituting metabolizable energy in cows during the early postpartum period suggest that supply of amino acids and glucogenic compounds may be under proposed optima, whereas ketogenic and lipogenic compounds and long-chain fatty acids may be in excess. Because dietary fat does not suppress body lipid mobilization, during the early post-partum period supplemental fat may further imbalance the mixture of fuels and lead to decreased dry matter intake. Increased understanding of the biology of the transition period should decrease health problems and increase profitability of dairy cows.
The onset of milk production lets mammals experience an enormous energy and nutrient demand. To meet these requirements, high-yielding dairy cows mobilize body fat resulting in an augmented hepatic oxidative metabolism, which has been suggested to signal for depressing hunger after calving. To examine how the extent of fat mobilization influences hepatic oxidative metabolism and thus potentially feed intake, blood and liver samples of 19 Holstein cows were taken throughout the periparturient period. Retrospectively grouped according to high (H) and low (L) liver fat content, H cows showed higher fatty acid but lower amino acid plasma concentrations and lower feed intake than L cows. The hepatic phospho-AMPK/total AMP ratio was not different between groups but decreased after parturition. A 2-DE coupled MALDI-TOF-TOF analysis and qRT-PCR studies revealed H cows having lower expressions of major enzymes involved in mitochondrial β-oxidation, urea cycling, and the pentose phosphate pathway but higher expressions of enzymes participating in peroxisomal and endoplasmic fatty acid degradation, pyruvate and TCA cycling, amino acid catabolism, oxidative phosphorylation, and oxidative stress defense. These data indicate that increasing lipolysis leads to augmenting nutrient catabolism for anaplerosis and mitochondrial respiration, providing a molecular link between hepatic oxidative processes and feed intake.
Heat stress (HT) during the dry period affects hepatic gene expression and adipose tissue mobilization during the transition period. In addition, it is postulated that HT may alter insulin action on peripheral tissues. Our objective was to evaluate the effect of cooling heat-stressed cows during the dry period on insulin effects on peripheral tissues during the transition period. Cows were dried off 46 d before expected calving and assigned to 1 of 2 treatments: HT (n = 16) or cooling (CL, n = 16). During the dry period, the average temperature-humidity index was 78, but CL cows were cooled with sprinklers and fans, whereas HT cows were not. After calving, all cows were housed and managed under the same conditions. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rate recorded 3 times weekly during the dry period. Dry matter intake was recorded daily from dry-off to 42 d relative to calving (DRC). Body weight and body condition score were measured weekly from dry-off to 42 DRC. Milk yield and composition were recorded daily to 42 wk postpartum. Glucose tolerance tests (GTT) and insulin challenges (IC) were performed at dry-off, -14, 7, and 28 DRC in a subset of cows (HT, n = 8; CL, n = 8). Relative to HT, CL cows had lower rectal temperatures (39.3 vs. 39.0°C) in the afternoon and respiration rate (69 vs. 48 breath/min). Cows from the cooling treatment tended to consume more feed than HT cows prepartum and postpartum. Compared with HT, CL cows gained more weight before calving but lost more weight and body condition in early lactation. Cows from the cooling treatment produced more milk than HT cows (34.0 vs. 27.7 kg/d), but treatments did not affect milk composition. Treatments did not affect circulating insulin and metabolites prepartum, but CL cows had decreased glucose, increased nonesterified fatty acid, and tended to have lower insulin concentrations in plasma postpartum compared with HT cows. Cooling prepartum HT cows did not affect the insulin responses to GTT and IC during the transition period and glucose responses to GTT and IC at -14 and 28 DRC were not affected by treatments. At 7 DRC, CL cows tended to have slower glucose clearance to GTT and weaker glucose response to IC relative to HT cows. Cows from the cooling treatment had stronger nonesterified fatty acid responses to IC postpartum but not prepartum compared with HT. In conclusion, cooling heat-stressed dairy cows in the dry period reduced insulin effects on peripheral tissues in early lactation but not in the dry period.
Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism.
Complex I deficiency is the most frequent cause of respiratory chain diseases. This large multiprotein complex is composed in human of 45 structural subunits, of which 7 are mitochondrial-encoded and 38 are nuclear-encoded. Most of the pathological mutations responsible for complex I deficiencies have been identified to date in complex I structural subunits. Numerous studies from last decade gave some insight into the biogenesis of this huge multi subunit complex of double genetic origin. A sequential incorporation of the structural subunits as well as ten complex I assembly factors has been described. Here, we present a short overview of the human complex I biogenesis and we review the pathological mutations identified to date in eight of the ten known complex I assembly factors.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that promotes the transcription of cytoprotective genes in response to oxidative and electrophilic stresses. Most functions of Nrf2 were identified by studying biological models with Nrf2 deficiency, however, little is known about the effects of graded Nrf2 activation. In the present study, genomic gene expression profiles by microarray analysis were characterized with a "gene dose-response" model in livers of Nrf2-null mice, wild-type mice, Kelch-like ECH associating protein 1 (Keap1)-knockdown (Keap1-KD) mice with enhanced Nrf2 activation, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum hepatic Nrf2 activation. Hepatic nuclear Nrf2 protein, glutathione concentrations, and known Nrf2 target genes were increased in a dose-dependent manner. In total, 115 genes were identified to be constitutively induced and 80 genes suppressed with graded Nrf2 activation. Messenger RNA of genes encoding enzymes in the pentose phosphate pathway and enzyme were low with Nrf2 deficiency and high with Nrf2 activation, indicating that Nrf2 is important for NADPH production. NADPH is the major reducing resource to scavenge oxidative stress, including regenerating glutathione and thioredoxin and is also used for anabolic pathways including lipid synthesis. High performance liquid chromatography-ultraviolet absorbance analysis confirmed that hepatic NADPH concentration was lowest in Nrf2-null mice and highest in Keap1-HKO mice. In addition, genes involved in fatty acid synthesis and desaturation were downregulated with graded Nrf2 activation. In conclusion, the present study suggests that Nrf2 protects against environmental insults by promoting the generation of NADPH, which is preferentially consumed by aiding scavenging of oxidative stress rather than fatty acid synthesis and desaturation.
Crohn's disease (CD) and ulcerative colitis (UC), known as inflammatory bowel disease (IBD), are fairly common chronic inflammatory conditions of the gastrointestinal tract. Although the exact etiology of IBD remains uncertain, dysfunctional immunoregulation of the gut is believed to be the main culprit. Amongst the immunoregulatory factors, reactive oxygen species are produced in abnormally high levels in IBD. Their destructive effects may contribute to the initiation and/or propagation of the disease. We provided an extensive overview on the evidences from animal and human literature linking oxidative stress to IBD and its activity. Moreover, the effects of antioxidant therapy on IBD patients in randomized, controlled trials were reviewed and the need for further studies elaborated. We also summarized the evidence in support for causality of oxidative stress in IBD.
peroxisome proliferator-activated receptor α (PPARα) is a crucial regulator of liver lipid metabolism. As maternal diabetes impairs foetal lipid metabolism and growth, we aimed to determine whether PPARα activation regulates lipid metabolism in the foetal liver from diabetic rats as well as foetal weight and foetal liver weight.
diabetes was induced by neonatal streptozotocin administration (90 mg/kg). For ex vivo studies, livers from 21-day-old foetuses from control and diabetic rats were explanted and incubated in the presence of PPARα agonists (clofibrate and leukotriene B(4) ) for further evaluation of lipid levels (by thin layer chromatography and densitometry), de novo lipid synthesis (by (14) C-acetate incorporation) and lipid peroxidation (by thiobarbituric reactive substances evaluation). For in vivo studies, foetuses were injected through the uterine wall with leukotriene B(4) on days 19, 20 and 21 of gestation. On day 21 of gestation, foetal liver concentrations of lipids and lipoperoxides were evaluated.
foetuses from diabetic rats showed increased body weight and liver weight, as well as accumulation of triglycerides and cholesteryl esters, increased de novo lipid synthesis and lipid peroxidation in the liver when compared to controls. Ex vivo studies showed that PPARα ligands reduced both the concentrations and synthesis of the lipid species studied and lipid peroxidation in the foetal liver from diabetic rats. In vivo experiments showed that leukotriene B(4) reduced the concentrations of triglycerides, cholesteryl esters and phospholipids, as well as lipid peroxidation, foetal weight and foetal liver weight in diabetic rats.
PPARα activation regulates the impaired foetal liver lipid metabolism, prevents hepatomegaly and reduces foetal overgrowth induced by maternal diabetes.
Heat stress (HT) and photoperiod affect milk production and immune status of dairy cows. The objective was to evaluate the effects of HT abatement prepartum under controlled photoperiod on hepatic metabolic gene expression and cellular immune function of periparturient Holstein cows (n=21). Cows were dried off 46 d before expected calving date and assigned to treatments by mature equivalent milk production. The treatments were 1) HT and 2) cooling (CL), both imposed during a photoperiod of 14L:10D. Rectal temperature was measured twice daily, whereas respiration rate was measured 3 times/wk at 1500 h during the entire dry period. After calving, cows were housed in a freestall barn with cooling, and milk yield was recorded daily up to 140 d in milk. Liver samples were taken at dry off, -20, 2, and 20 d relative to calving by biopsy. Under a similar schedule, neutrophil function was determined in blood of cows on HT (n=12) and CL (n=9). Blood samples were taken on -46, -32, -18, 0, 14, 28, and 42 d relative to calving for measurement of metabolites and were collected twice daily from -7 to 2 d relative to calving for prolactin (PRL) analysis. The HT cows had greater concentrations of PRL at 0 d relative to calving (150 vs. 93; SEM=11 ng/mL) and had higher afternoon rectal temperatures (39.4 vs. 39.0; SEM=0.04°C) and elevated respiration rates (78 vs. 56; SEM=2 breaths/min) during the prepartum period compared with CL cows. Relative to HT cows, CL cows had greater hepatic expression of PRL-R, SOCS-3, and CAV-1 mRNA. Neutrophil oxidative burst was greater in CL cows relative to HT cows at 2 d (61 vs. 42; SEM=6%) and at 20 d (62 vs. 49; SEM=5%) relative to calving, and phagocytosis was greater in CL cows at 20 d (47 vs. 33; SEM=4%) relative to calving compared with HT cows. Humoral response, as measured by IgG secretion against ovalbumin challenge, was greater for CL cows at -32 d (0.44 vs. 0.33; SEM=0.05 OD) and -21 d (0.60 vs. 0.50±0.04 OD) relative to calving compared with HT cows. These results suggest that HT abatement during the dry period improved innate and acquired immune status as measured by neutrophil function and immunoglobulin secretion against ovalbumin challenge, and altered hepatic gene expression related to PRL signaling in the periparturient period or subsequent lactation.
Dairy cows undergo tremendous metabolic and physiological adaptations around parturition to support lactation. The liver is central to many of these processes, including gluconeogenesis and metabolism of fatty acids mobilized from adipose tis- sue. Fat accumulation may impair normal functions of the liver and increase ketogenesis, which in turn may predispose cows to other metabolic abnormalities. Several aspects of dietary management and body condition may alter these adaptations, affect dry matter intake, and increase or decrease susceptibility to periparturient health problems. Overfeeding energy dur- ing the dry period is a prominent risk factor. Considerable progress has been made in recent years in describing the adap- tive changes in the liver and other organs in normal and abnormal states, but this knowledge has not yet identified unequiv- ocally the key steps that might be compromised during development of metabolic disorders. The potential role of signaling compounds, such as the inflammatory cytokines released in response to environmental stressors, infectious challenge, and oxidative stress, in the pathogenesis of periparturient disease is under investigation. New techniques such as functional genomics, using cDNA or oligonucleotide microarrays, as well as proteomics and metabolomics, provide additional high- throughput tools to determine the effects of nutrition, management, or stressors on tissue function in development of dis- ease. Integrative approaches should be fruitful in unraveling the complex interactions of metabolism, immune activation, stress physiology, and endocrinology that likely underlie development of periparturient disease.
Environmental factors, especially temperature and light exposure, influence the health and productivity of dairy cows during lactation, possibly via similar physiological mechanisms. For example, heat stress is a critical component of decreased milk yield during summer. However, less is known about the effect of heat stress during the dry period. The objective of this study was to evaluate the effects of heat stress prepartum under a controlled photoperiod on lactation performance and hepatic metabolic gene expression of periparturient multiparous Holstein cows (n = 16). Cows were dried off approximately 46 d before expected calving date and assigned to treatment randomly after blocking by mature equivalent milk production and parity. Treatments consisted of either heat stress (HT) or cooling (CL) with fans and sprinklers, both under a photoperiod of 14L:10D. Rectal temperature was measured twice daily during the dry period. After calving, cows were housed in a freestall barn with cooling devices, and milk yield was recorded daily up to 210 d in milk. Blood samples were taken from dry off until +42 d relative to calving for metabolites and from -2 until +2 d relative to calving for hormone analysis. Daily dry matter intake was measured from -35 to +42 d relative to calving. Liver biopsies were collected at dry off, -20, +2, and +20 d relative to calving for cows on HT (n = 5) and CL (n = 4) to measure mRNA expression of suppressors of cytokine signaling-2 (SOCS-2), insulin-like growth factor binding protein-5 (IGFBP-5), a key transcription factor in lipid biosynthesis (SREBP-1c), and enzymes of lipid metabolism (FASN, ACACA, and ACADVL) by real-time quantitative PCR. Heat stress increased rectal temperatures (39.2 vs. 38.8 degrees C), plasma prolactin concentrations at -1 (171 vs. 79 ng/mL) and 0 d (210 vs. 115 ng/mL) relative to calving, and decreased dry matter intake at 0 and +14 d relative to calving and 3.5% fat-corrected milk postpartum (26.1 vs. 35.4 kg/d) compared with CL cows. Relative to CL cows, hepatic mRNA expression of SOCS-2 and IGFBP-5 was downregulated in HT cows. Expression of ACADVL was upregulated in CL cows at d +2 but downregulated at d +20 relative to HT cows. Concentrations of C16:0 and cis C18:1 were greater in the milk and liver of CL cows compared with HT cows, which reflects greater lipid mobilization. These results suggest that heat-stress abatement in the dry period improves subsequent lactation, possibly via suppression of plasma prolactin surge around calving, SOCS-2 expression, and regulation of hepatic lipid metabolism.
We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry-based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
The inner membranes of mitochondria contain three multi-subunit enzyme complexes that act successively to transfer electrons from NADH to oxygen, which is reduced to water (Fig. I). The first enzyme in the electron transfer chain, NADH:ubiquinone oxidoreductase (or complex I), is the subject of this review. It removes electrons from NADH and passes them via a series of enzyme-bound redox centres (FMN and Fe-S clusters) to the electron acceptor ubiquinone. For each pair of electrons transferred from NADH to ubiquinone it is usually considered that four protons are removed from the matrix (see section 4.1 for further discussion of this point).
The primary source of fatty acids processed by ruminant liver is nonesterified fatty acids (NEFA) from blood. Uptake is regulated by concentration of NEFA and blood flow. Blood NEFA concentration increases with negative energy balance. Blood flow increases with energy intake. Uptake and secretion of triacylglycerol between blood and the liver is limited. The reason for limited hepatic secretion of triacylglycerol-rich lipoprotein is unclear but probably involves the secretory process, not synthesis of triacylglycerol or apolipoprotein. Oxidation of fatty acids and ketogenesis are inhibited by malonyl-CoA and propionic acid. The stress of late gestation and early lactation increases NEFA supply to the liver, where they cause deposition of fat. Ketogenesis and oxidation in the liver increase but not sufficiently to prevent an accumulation of fat, which may contribute to decreased feed intake in the peripartum period.
For an evaluation of the effects of heat stress during pregnancy on calf birth weight and postpartum maternal milk yield, 21 cows and 10 heifers were assigned to shade (S; n = 16) or no shade (NS; n = 15) treatments during the last trimester of pregnancy in June 1978. At parturition, all cows were removed from treatment and uniformly managed in the milking herd. At 4-d intervals from d 199 of pregnancy to parturition, respiration rates, rectal temperature, heart rate and Black Globe temperature were recorded and blood samples obtained via tail venipuncture between 1300 and 1600 h. Weekly prepartum body weights of dam, birth weight of calf and subsequent daily milk yield were recorded. Black Globe temperature, rectal temperature and respiration rates were higher in cows given NS. Calf birth weight was lower in the NS group. Milk yield was correlated in a linear manner with calf birth weight, and cows in group NS exhibited reduced lactation performance after calving. Plasma progestin concentrations were higher in heat-stressed cows (6.0 vs 5.1 ng/ml). Estrone-sulfate concentrations were reduced in plasma of NS cows (2,505 vs 4,433 pg/ml). Thus, hormone concentrations of maternal and fetal origin were altered by environment. Plasma thyroxine concentrations were lower in NS cows (51.2 vs 66.4 ng/ml), while plasma triiodothyronine concentrations were elevated (1.8 vs 1.5 ng/ml), indicating altered thyroid hormone metabolism in heat-stressed cows. Heat stress altered endocrine dynamics during pregnancy and reduced calf birth weight and may have indirectly altered subsequent milk yield.
Most of the metabolic diseases of dairy cows-milk fever, ketosis, retained placenta, and displacement of the abomasum-occur within the first 2 wk of lactation. The etiology of many of those metabolic diseases that are not clinically apparent during the first 2 wk of lactation, such as laminitis, can be traced back to insults that occurred during early lactation. In addition to metabolic disease, the overwhelming majority of infectious disease, in particular mastitis, becomes clinically apparent during the first 2 wk of lactation. Three basic physiological functions must be maintained during the periparturient period if disease is to be avoided: adaptation of the rumen to lactation diets that are high in energy density, maintenance of normocalcemia, and maintenance of a strong immune system. The incidence of both metabolic and infectious diseases is greatly increased whenever one or more of these physiological functions are impaired. This paper discusses the etiological role of each of these factors in the development of common diseases encountered during the periparturient period.
Iron is required for normal cell growth and proliferation. However, excess iron is potentially harmful, as it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton chemistry. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels. Chief among these is the sequestration of iron in ferritin. Ferritin is a 24 subunit protein composed of two subunit types, termed H and L. The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. In the present study we report that increased synthesis of both subunits of ferritin occurs in HeLa cells exposed to oxidative stress. An increase in the activity of iron responsive element binding proteins in response to oxidative stress was also observed. However, this activation was transient, allowing ferritin protein induction to subsequently proceed. To assess whether ferritin induction reduced the accumulation of ROS, and to test the relative contribution of ferritin H and L subunits in this process, we prepared stable transfectants that overexpressed either ferritin H or ferritin L cDNA under control of a tetracycline-responsive promoter. We observed that overexpression of either ferritin H or ferritin L reduced the accumulation of ROS in response to oxidant challenge.
At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, however, nitric oxide (NO), superoxide anion, and related reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and reestablish "redox homeostasis." Higher organisms, however, have evolved the use of NO and ROS also as signaling molecules for other physiological functions. These include regulation of vascular tone, monitoring of oxygen tension in the control of ventilation and erythropoietin production, and signal transduction from membrane receptors in various physiological processes. NO and ROS are typically generated in these cases by tightly regulated enzymes such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. In a given signaling protein, oxidative attack induces either a loss of function, a gain of function, or a switch to a different function. Excessive amounts of ROS may arise either from excessive stimulation of NAD(P)H oxidases or from less well-regulated sources such as the mitochondrial electron-transport chain. In mitochondria, ROS are generated as undesirable side products of the oxidative energy metabolism. An excessive and/or sustained increase in ROS production has been implicated in the pathogenesis of cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases, rheumatoid arthritis, ischemia/reperfusion injury, obstructive sleep apnea, and other diseases. In addition, free radicals have been implicated in the mechanism of senescence. That the process of aging may result, at least in part, from radical-mediated oxidative damage was proposed more than 40 years ago by Harman (J Gerontol 11: 298-300, 1956). There is growing evidence that aging involves, in addition, progressive changes in free radical-mediated regulatory processes that result in altered gene expression.