Cardiac-specific overexpression of thioredoxin 1 attenuates mitochondrial and myocardial dysfunction in septic mice

Sepsis-induced myocardial dysfunction is associated with increased oxidative stress and mitochondrial dysfunction. Current evidence suggests a protective role of thioredoxin-1 (Trx1) in the pathogenesis of cardiovascular diseases. However, it is unknown yet a putative role of Trx1 in sepsis-induced myocardial dysfunction, in which oxidative stress is an underlying cause. Transgenic male mice with Trx1 cardiac-specific overexpression (Trx1-Tg) and its wild-type control (wt) were subjected to cecal ligation and puncture or sham surgery. After 6, 18, and 24 h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28 h; p = 0.0204). Sepsis induced a decrease in left ventricular developed pressure in both groups, while the contractile reserve, estimated as the response to β-adrenergic stimulus, was higher in Trx1-Tg in relation to wt, after 6 h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24 h. Ultrastructural alterations in mitochondrial cristae were accompanied by reduced optic atrophy 1 (OPA1) fusion protein, and activation of dynamin-related protein 1 (Drp1) (fission protein) in wt mice at 24 h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24 h, suggesting mitochondrial biogenesis induction. Autophagy, demonstrated by electron microscopy and increased LC3-II/LC3-I ratio, was observed earlier in Trx1-Tg. In conclusion, Trx1 overexpression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover (mitophagy and biogenesis), preserves contractile reserve and prolongs survival during sepsis.     

Mitofusin 2 ameliorates hypoxia-induced apoptosis via mitochondrial function and signaling pathways

Mitochondrial dynamics play a critical role in mitochondrial function and signaling. Although mitochondria play a critical role in hypoxia/ischemia, the further mechanisms between mitochondrial dynamics and ischemia are still unclear. The current study aimed to determine the role of mitofusin 2, a key regulator of mitochondrial fusion, in a hypoxic model and to explore a novel strategy for cerebral ischemia via modulation of mitochondrial dynamics. To the best of our knowledge, this is the first study to investigate both mitochondrial function and molecular pathways to determine the role of mitofusin 2 in hypoxia-induced neuronal apoptosis. In vivo, C57BL/6 mice (male, 19–25 g) underwent a permanent middle cerebral artery occlusion for 12 or 24 h (n = 6 per group). In vitro, cobalt chloride was used to mimic hypoxia in immortalized hippocampal neurons. Down- or up-regulation of Mfn2 was induced to investigate the role of Mfn2 in hypoxia, especially in mitochondrial function and signaling pathways. The findings demonstrated that decreased mitofusin 2 occurred both in vivo and in vitro hypoxic models; second, the anti-apoptotic effect of Mfn2 may work via restoration of mitochondrial function; third, the modulation of the B Cell Leukemia 2/Bcl-2 Associated X protein and extracellular signal-regulated kinase 1/2 signaling pathways highlight the role of Mfn2 in signaling pathways beyond fusion. In summary, depletion of mitofusin 2 would lead to apoptosis both in normal or hypoxic conditions; however, mitofusin 2 overexpression could attenuate hypoxia-induced apoptosis, which represents a potential novel strategy for neuroprotection against ischemic brain damage.     

Diallyl disulfide ameliorates isoproterenol induced cardiac hypertrophy activating mitochondrial biogenesis via eNOS-Nrf2-Tfam pathway in rats

The beneficial effect of garlic on cardiovascular disease is well known. However, the use of raw garlic against cardiac hypertrophy is not established. In the present study we explored whether raw garlic and its compound, diallyl disulfide (DADS) could inhibit hypertrophy through H₂S and/or mitochondrial biogenesis. Cardiac hypertrophy was induced in rat by giving isoproterenol at the dose of 5 mg/kg/day subcutaneously for 14 days through alzet minipump. Aqueous garlic homogenate, DADS and NaHS (liberate H₂S) were fed to third, forth and fifth group of rats for 14 days at a dose of 250 mg/kg/day, 50 mg/kg/day, 14 µM/kg/day respectively. Garlic and DADS reduced cardiac hypertrophy markers and normalized mitochondrial ETC-complex activities, mitochondrial enzyme activites and mitochondrial biogenetic and apoptotic genes expression. Garlic and DADS enhanced eNOS and p-AKT level in rat heart along with increased NRF2 protein level and Tfam gene expression. However, normalization was not observed after administration of NaHS which generates H₂S in-vivo. In conclusion, garlic and DADS induces mitochondrial biogenesis and ameliorates cardiac hypertrophy via activation of eNOS-Nrf2-Tfam pathway in rats.     

Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.)

To test the hypothesis that impaired mitochondrial respiration limits cardiac performance at warm temperatures, and examine if any effect(s) are sex-related, the consequences of high temperature on cardiac mitochondrial oxidative function were examined in 10 °C acclimated, sexually immature, male and female Atlantic cod. Active (State 3) and uncoupled (States 2 and 4) respiration were measured in isolated ventricular mitochondria at 10, 16, 20, and 24 °C using saturating concentrations of malate and pyruvate, but at a submaximal (physiological) level of ADP (200 µM). In addition, citrate synthase (CS) activity was measured at these temperatures, and mitochondrial respiration and the efficiency of oxidative phosphorylation (P:O ratio) were determined at [ADP] ranging from 25–200 µM at 10 and 20 °C. Cardiac morphometrics and mitochondrial respiration at 10 °C, and the thermal sensitivity of CS activity (Q₁₀=1.51), were all similar between the sexes. State 3 respiration at 200 µM ADP increased gradually in mitochondria from females between 10 and 24 °C (Q₁₀=1.48), but plateaued in males above 16 °C, and this resulted in lower values in males vs. females at 20 and 24 °C. At 10 °C, State 4 was ~10% of State 3 values in both sexes [i.e. a respiratory control ratio (RCR) of ~10] and P:O ratios were approximately 1.5. Between 20 and 24 °C, State 4 increased more than State 3 (by ~70 vs. 14%, respectively), and this decreased RCR to ~7.5. The P:O ratio was not affected by temperature at 200 μM ADP. However, (1) the sensitivity of State 3 respiration to increasing [ADP] (from 25 to 200 μM) was reduced at 20 vs. 10 °C in both sexes (K_m values 105±7 vs. 68±10 μM, respectively); and (2) mitochondria from females had lower P:O values at 25 vs. 100 μM ADP at 20 °C, whereas males showed a similar effect at 10 °C but a much more pronounced effect at 20 °C (P:O 1.05 at 25 μM ADP vs. 1.78 at 100 μM ADP). In summary, our results demonstrate several sex-related differences in ventricular mitochondrial function in Atlantic cod, and suggest that myocardial oxidative function and possibly phosphorylation efficiency may be limited at temperatures of 20 °C or above, particularly in males. These observations could partially explain why cardiac function in Atlantic cod plateaus just below this species׳ critical thermal maximum (~22 °C) and may contribute to yet unidentified sex differences in thermal tolerance and swimming performance.     

Temperature-related differences in mitochondrial function among clones of the cladoceran Daphnia pulex

This study assessed the thermal sensitivity of mitochondrial respiration in the small crustacean Daphnia pulex. More specifically, we wanted to determine if clones that inhabit different latitudes and habitats showed differences in the thermal sensitivity of their mitochondrial function. The experimental design included two clones from temperate environments (Fence from Ontario and Hawrelak from Alberta) and two clones from subarctic environments (A24 from Manitoba and K154 from Quebec). The integrated mitochondrial function was measured with high-resolution respirometry following whole-animal permeabilization. Mitochondrial respiration was performed under six different temperatures (10, 15, 20, 25, 30, and 35 °C) in the clone Hawrelak and at two temperatures (10 and 20 °C) in the three other clones. In the clone Hawrelak, complexes I and II respiration showed higher sensitivity to temperature variation compared to complex IV respiration. Interestingly, the threshold plot showed no excess capacity of complex IV at 20 °C in this clone. The clones showed significant divergence in the ability to oxidize the complex I and complex IV substrates relative to the maximal oxidative phoshorylation capacity of mitochondria. More importantly, some of the clonal divergences were only detected under low assay temperatures, pointing toward the importance of this parameter in comparative studies. Future and more complex studies on clones from wider environmental gradients will help to resolve the link between mitochondrial function and adaptations of organisms to particular conditions, principally temperature.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Vasoactive intestinal peptide (VIP) differentially affects inflammatory immune responses in human monocytes infected with viable Salmonella or stimulated with LPS

We compared the effect of VIP on human blood monocytes infected with Salmonella typhimurium 4/74 or stimulated with LPS. VIP (10⁻⁷ M) increased monocyte viability by 24% and 9% when cultured for 24 h with 4/74 or Salmonella LPS (100 ng/ml), respectively. Significantly increased (P < 0.05) numbers of 4/74 were also recovered from monocytes co-cultured with VIP after 6 h post-infection (pi) and this remained high after 24 h pi. Both 4/74 and LPS increased (P < 0.05) the concentration of TNF-α, IL-1β and IL-6 measured in monocyte supernatants. However, LPS induced this effect more rapidly while, with the exception of IL-6, 4/74 induced higher concentrations (P < 0.05). VIP significantly decreased (P < 0.05) TNF-α and IL-1β production by 4/74-infected monocytes after 6 pi, but only after 24 h in LPS-cultured monocytes. This trend was reversed for IL-6 production. However, TNF-α and IL-1β production by 4/74-infected monocytes, cultured with VIP, still remained higher (P < 0.05) than concentrations measured in supernatants cultured only with LPS. VIP also increased (P < 0.05) production of anti-inflammatory IL-10 in both 4/74 and LPS cultures after 24 h. We also show a differential effect of VIP on the expression of TNFα and IL-6 receptors, since VIP was only able to decreased expression in LPS-stimulated monocytes but not in 4/74-infected monocytes.In conclusion, we show a differential effect of VIP on human monocytes infected with virulent Salmonella or stimulated with LPS. Our study suggests that the use of VIP in bacteraemia and/or sepsis may be limited to an adjunctive therapy to antibiotic treatment.     

MITOCHONDRIA: Investigation of in vivo muscle mitochondrial function by 31P magnetic resonance spectroscopy

The most important function of mitochondria is the production of energy in the form of ATP. The socio-economic impact of human diseases that affect skeletal muscle mitochondrial function is growing, and improving their clinical management critically depends on the development of non-invasive assays to assess mitochondrial function and monitor the effects of interventions. ³¹P magnetic resonance spectroscopy provides two approaches that have been used to assess in vivo ATP synthesis in skeletal muscle: measuring P_i ⟶ ATP exchange flux using saturation transfer in resting muscle, and measuring phosphocreatine recovery kinetics after exercise. However, P_i ⟶ ATP exchange does not represent net mitochondrial ATP synthesis flux and has no simple relationship with mitochondrial function. Post-exercise phosphocreatine recovery kinetics, on the other hand, yield reliable measures of muscle mitochondrial capacity in vivo, whose ability to define the site of functional defects is enhanced by combination with other non-invasive techniques.     

Cajanol,a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots,induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (ΔΨm) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC₅₀ value was 54.05 μM after 72 h treatment, 58.32 μM after 48 h; and 83.42 μM after 24 h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro.     

The mitochondrial calcium uniporter is involved in mitochondrial calcium cycle dysfunction: Underlying mechanism of hypertension associated with mitochondrial tRNAIle A4263G mutation

Recent studies have shown that the mitochondrial DNA mutations are involved in the pathogenesis of hypertension. Our previous study identified mitochondrial tRNA^Ile A4263G mutation in a large Chinese Han family with maternally-inherited hypertension. This mutation may contribute to mitochondrial Ca²⁺ cycling dysfuntion, but the mechanism is unclear. Lymphoblastoid cell lines were derived from hypertensive and normotensive individuals, either with or without tRNA^Ile A4263G mutation. The mitochondrial calcium ([Ca²⁺]_m) in cells from hypertensive subjects with the tRNA^Ile A4263G mutation, was lower than in cells from normotension or hypertension without mutation, or normotension with mutation (P < 0.05). Meanwhile, cytosolic calcium ([Ca²⁺]_c) in hypertensive with mutation cells was higher than another three groups. After exposure to caffeine, which could increase the [Ca²⁺]_c by activating ryanodine receptor on endoplasmic reticulum, [Ca²⁺]_c/[Ca²⁺]_m increased higher than in hypertensive with mutation cells from another three groups. Moreover, MCU expression was decreased in hypertensive with mutation cells compared with in another three groups (P < 0.05). [Ca²⁺]_c increased and [Ca²⁺]_m decreased after treatment with Ru360 (an inhibitor of MCU) or an siRNA against MCU. In this study we found decreased MCU expression in hypertensive with mutation cells contributed to dysregulated Ca²⁺ uptake into the mitochondria, and cytoplasmic Ca²⁺ overload. This abnormality might be involved in the underlying mechanisms of maternally inherited hypertension in subjects carrying the mitochondrial tRNA^Ile A4263G mutation.     

Rapid cloning,expression and purification of a novel high-activity alkaline phosphatase with detoxification of lipopolysaccharide

Lipopolysaccharide (LPS) is a bacterial endotoxin leading to endotoxemia. Its virulence factor ‘diphosphoryl lipid A’ can be abolished by alkaline phosphatase (AP). A novel AP gene (without introns) was cloned from Saccharomyces boulardii ATCC MYA-796 with a GenBank accession number KF471017, and the recombinant AP (rAP) was expressed as a soluble protein in Pichia pastoris X-33 with a yield of 43.66 mg/l at the end of 120 h of induction in a shaker flask. After purification by affinity-column chromatography, the purity of rAP was over 90%. The optimal reaction conditions of rAP were pH 9.6, temperature at 60 °C and 2 mM Mg²⁺ in diethanolamine buffer, and EDTA was a potent inhibitor of rAP activity. The specific activity of rAP was 9912.01 U/mg under the optimal conditions. Furthermore, rAP showed a broad dephosphorylation activity to LPS over a broad pH range (pH 2–10) in vitro and peaked at pH 4 in Tris–HCl buffer. After LPS dephosphorylated by rAP was injected intraperitoneally into mice, the serum level of tumor necrosis factor (TNF)-α was significantly reduced compared to that of the LPS group (p < 0.01). These findings suggest that rAP has great potential to cure diseases caused by LPS.     

Upregulation of autophagy decreases chlorine-induced mitochondrial injury and lung inflammation

The mechanisms of toxicity during exposure of the airways to chlorinated biomolecules generated during the course of inflammation and to chlorine (Cl₂) gas are poorly understood. We hypothesized that lung epithelial cell mitochondria are damaged by Cl₂ exposure and activation of autophagy mitigates this injury. To address this, NCI-H441 (human lung adenocarcinoma epithelial) cells were exposed to Cl₂ (100 ppm/15 min) and bioenergetics were assessed. One hour after Cl₂, cellular bioenergetic function and mitochondrial membrane potential were decreased. These changes were associated with increased MitoSOX signal, and treatment with the mitochondrial redox modulator MitoQ attenuated these bioenergetic defects. At 6 h postexposure, there was significant increase in autophagy, which was associated with an improvement of mitochondrial function. Pretreatment of H441 cells with trehalose (an autophagy activator) improved bioenergetic function, whereas 3-methyladenine (an autophagy inhibitor) resulted in increased bioenergetic dysfunction 1 h after Cl₂ exposure. These data indicate that Cl₂ induces bioenergetic dysfunction, and autophagy plays a protective role in vitro. Addition of trehalose (2 vol%) to the drinking water of C57BL/6 mice for 6 weeks, but not 1 week, before Cl₂ (400 ppm/30 min) decreased white blood cells in the bronchoalveolar lavage fluid at 6 h after Cl₂ by 70%. Acute administration of trehalose delivered through inhalation 24 and 1 h before the exposure decreased alveolar permeability but not cell infiltration. These data indicate that Cl₂ induces bioenergetic dysfunction associated with lung inflammation and suggests that autophagy plays a protective role.     

Diabetes impairs heart mitochondrial function without changes in resting cardiac performance

Diabetes is a chronic disease associated to a cardiac contractile dysfunction that is not attributable to underlying coronary artery disease or hypertension, and could be consequence of a progressive deterioration of mitochondrial function. We hypothesized that impaired mitochondrial function precedes Diabetic Cardiomyopathy. Thus, the aim of this work was to study the cardiac performance and heart mitochondrial function of diabetic rats, using an experimental model of type I Diabetes. Rats were sacrificed after 28 days of Streptozotocin injection (STZ, 60 mg kg⁻¹, ip.). Heart O₂ consumption was declined, mainly due to the impairment of mitochondrial O₂ uptake. The mitochondrial dysfunction observed in diabetic animals included the reduction of state 3 respiration (22%), the decline of ADP/O ratio (∼15%) and the decrease of the respiratory complexes activities (22–26%). An enhancement in mitochondrial H₂O₂ (127%) and NO (23%) production rates and in tyrosine nitration (58%) were observed in heart of diabetic rats, with a decrease in Mn-SOD activity (∼50%). Moreover, a decrease in contractile response (38%), inotropic (37%) and lusitropic (58%) reserves were observed in diabetic rats only after a β‐adrenergic stimulus. Therefore, in conditions of sustained hyperglycemia, heart mitochondrial O₂ consumption and oxidative phosphorylation efficiency are decreased, and H₂O₂ and NO productions are increased, leading to a cardiac compromise against a work overload. This mitochondrial impairment was detected in the absence of heart hypertrophy and of resting cardiac performance changes, suggesting that mitochondrial dysfunction could precede the onset of diabetic cardiac failure, being H₂O₂, NO and ATP the molecules probably involved in mitochondrion-cytosol signalling.     

The effect of treadmill training and N-acetyl-l-cysteine intervention on biogenesis of cytochrome c oxidase (COX)

Mitochondrial biogenesis refers to increased content of mitochondria, which has been shown to be promoted by aerobic exercise. During this process, oxidative stress is considered the essential initiator. Even though some studies have addressed the issue as to whether antioxidants would hamper the effects of exercise on mitochondrial biogenesis, no consensus has been achieved. Therefore, the purpose of the present study was to investigate the effects of exercise and antioxidant intervention on mitochondrial biogenesis, as well as COX biogenesis. Thirty-two clean-grade male ICR mice were randomly assigned to a control group (Con), exercise group (Ex), N-acetyl-l-cysteine group (NAC), or NAC plus exercise group (NEx). The NAC and NEx groups were injected with NAC (0.1 mg/g/2 days) intraperitoneally for 3 weeks, whereas the Con and Ex groups were administered saline for the same period of time. Mice assigned to Ex and NEx groups started exercise training 1 week before drug intervention was initiated. After 1 week of acclimatization, the mice were allowed to run at a speed of 28 m/min for 60 min, 6 days a week. The results showed that exercise training caused an increase in mRNA and protein levels of COXIV, whereas NAC intervention lowered the two so significantly that even exercise training could not reverse the effect of NAC intervention. Our data suggest that even though antioxidant intervention could alleviate oxidative damage caused by exercise, it was not necessarily beneficial for mitochondrial biogenesis.     

Local muscle cooling does not impact expression of mitochondrial-related genes

Recovery that takes place in a cold environment after endurance exercise elevates PGC-1α mRNA whereas ERRα and NRF2 mRNA expression are inhibited. However, the effect of local skeletal muscle cooling on mitochondrial-related gene expression is unknown.PurposeTo determine the impact of local skeletal muscle cooling during recovery from an acute bout of exercise on mitochondrial-related gene expression.MethodsRecreationally-trained male cyclists (n=8, age 25±3 y, height 181±6 cm, weight 79±8 kg, 12.8±3.6% body fat, VO_2peak 4.52±0.88 L·min⁻¹ protocol) completed a 90-min variable intensity cycling protocol followed by 4 h of recovery. During recovery, ice was applied intermittently to one leg (ICE) while the other leg served as a control (CON). Intramuscular temperature was recorded continuously. Muscle biopsies were taken from each vastus lateralis at 4 h post-exercise for the analysis of mitochondrial-related gene expression.ResultsIntramuscular temperature was colder in ICE (26.7±1.1 °C) than CON (35.5±0.1 °C) throughout the 4 h recovery period (p<0.001). There were no differences in expression of PGC-1α, TFAM, NRF1, NRF2, or ERRα mRNA between ICE and CON after the 4 h recovery period.ConclusionLocal muscle cooling after exercise does not impact the expression of mitochondrial biogenesis-related genes compared to recovery from exercise in control conditions. When these data are considered with previous research, the stimuli for cold-induced gene expression alterations may be related to factors other than local muscle temperature. Additionally, different intramuscular temperatures should be examined to determine dose-response of mitochondrial-related gene expression.     

Effect of dietary β-glucan on growth performance,fecal microbial shedding and immunological responses after lipopolysaccharide challenge in weaned pigs

Two experiments (Exp.) were conducted to evaluate the effects of β-glucan inclusion in the diet on growth performance and immune function after lipopolysaccharide (LPS) challenge. In Exp. 1, a total of 40 weaned pigs (progeny of Landrace×Yorkshire sows by Duroc) with an initial body weight (BW) of 7.89 ± 0.84 kg (21 ± 2 d) of age) were used in a 28-day (d) experiment to determine the effects of dietary β-glucan on growth performance. Pigs were allotted randomly to two treatments consisting of addition of 0 or 0.1 g β-glucan/kg diet with four replicate pens per treatment and five pigs per pen. Growth performance was not affected by β-glucan supplementation throughout the experiment. However, dietary β-glucan reduced (P<0.05) the number of fecal Escherichia coli. In Exp. 2, a total of 20 weaned barrows (6.22 ± 0.25 kg of BW and 21 ± 2 d of age) individually raised in metabolic cages were used to evaluate immunological responses following LPS challenge. Pigs were fed 0 or 0.1 g β-glucan/kg diet for 42 d. At the end of the trial, half of the pigs (n = 5) from each treatment were injected intraperitoneal with E. coli LPS at a concentration of 100 μg/kg BW and the other half were injected with sterile saline solution. Treatments were arranged as a 2×2 factorial, with the main effect of LPS challenge (saline vs. LPS) and β-glucan supplementation (0 g/kg vs. 0.1 g/kg). After LPS injection, blood was taken at 0, 2, 4, 6, 8 and 12 hours (h) for the blood cell counts and blood inflammatory response. Dietary β-glucan increased (P<0.05) leukocytes counts at 4, 6 and 8 h, and blood lymphocyte concentrations at 2, 4 and 6 h and LPS challenge increased (P<0.05) counts of leukocytes at 2, 4, 6 and 8 h and blood lymphocyte at 2 and 4 h post-challenge. The rectal temperature was increased (P<0.05) at 2, 4, 6 and 8 h after LPS challenge. Dietary β-glucan reduced (P<0.05) and LPS challenge increased (P<0.05) blood plasma tumor necrosis factor-α (TNF-α) concentration at 2 and 4 h post-challenge. Dietary β-glucan increased (P<0.05) the concentration of the cluster of differentiation antigens 4 cells (CD4⁺) at 2, 4 and 6 h, and of 8 (CD8⁺) at 4 and 6 h post-challenge, respectively. The LPS challenge increased (P<0.05) CD4⁺ and CD8⁺ cell concentrations at 2, 4 and 6 h post-challenge. The CD4⁺:CD8⁺ ratio was reduced (P<0.05) by LPS challenge but was increased (P<0.05) by dietary β-glucan at 2, 4, 6 and 8 h post-challenge. In conclusion, dietary β-glucan decreased E. coli numbers but did not affect growth performance in weaned pigs and may offer benefits on immune function in weaned pigs challenged with LPS.     

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: Involvement of mitochondrial aquaporin-8

We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15 N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis.