Liaman Mamedova

Analysis of rumen microbial populations in lactating dairy cattle fed diets varying in carbohydrate profiles and Saccharomyces cerevisiae fermentation product

The rumen microbial ecosystem is a critical factor that links diets to bovine physiology and productivity; however, information about dietary effects on microbial populations has generally been limited to small numbers of samples and qualitative assessment. To assess whether consistent shifts in microbial populations occur in response to common dietary manipulations in dairy cattle, samples of rumen contents were collected from 2 studies for analysis by quantitative real-time PCR (qPCR). In one study, lactating Holstein cows (n=8) were fed diets in which a nonforage fiber source replaced an increasing proportion of forages and concentrates in a 4×4 Latin square design, and samples of ruminal digesta were collected at 9-h intervals over 3 d at the end of each period. In the second study, lactating Holstein cows (n=15) were fed diets with or without the inclusion of a Saccharomyces cerevisiae fermentation product (SCFP) in a crossover design. In this study, rumen liquid and solid samples were collected during total rumen evacuations before and after feeding in a 42-h period. In total, 146 samples of ruminal digesta were used for microbial DNA isolation and analysis by qPCR. Validated primer sets were used to quantify total bacterial and anaerobic fungal populations as well as 12 well-studied bacterial taxa. The relative abundance of the target populations was similar to those previously reported. No significant treatment effects were observed for any target population. A significant interaction of treatment and dry matter intake was observed, however, for the abundance of Eubacterium ruminantium. Increasing dry matter intake was associated with a quadratic decrease in E. ruminantium populations in control animals but with a quadratic increase in E.ruminantium populations in cows fed SCFP. Analysis of sample time effects revealed that Fibrobacter succinogenes and fungal populations were more abundant postfeeding, whereas Ruminococcus albus tended to be more abundant prefeeding. Seven of the target taxa were more abundant in either the liquid or solid fractions of ruminal digesta. By accounting for the total mass of liquid and solid fractions in the rumen and the relative abundance of total bacteria in each fraction, it was estimated that 92% of total bacteria were found in the solid digesta fraction.

An unusual distribution of the niacin receptor in cattle.

Responses to pharmacological doses of niacin, an agonist for GPR109A (niacin receptor), were different in cattle than in humans and rodents. Thus, the tissue distribution of GPR109A was investigated in cattle. Samples of tail head fat, back fat, perirenal fat, longissimus muscle, and liver were analyzed for abundance of GPR109A mRNA by quantitative real-time reverse transcription-PCR and for abundance of GPR109A protein by Western blotting. Niacin receptor transcript and protein were detected in all tissues analyzed. The mRNA for GPR109A was more abundant in liver than in the other tissues sampled (GPR109A:RPS9 mRNA abundance = 0.56 in liver compared with 0.06 in longissimus muscle, 0.15 in kidney fat, 0.11 in back fat, 0.23 in tail head fat; standard error of the mean = 0.028). Additionally, mRNA for GPR109A was found (GPR109A:RPS9 mRNA abundance ≥ 0.004) in each of the 5 regions of bovine brain that were analyzed: cerebral cortex, cerebellum, thalamus, hypothalamus, and brain stem. Evaluation of liver tissue by immunofluorescence suggested that GPR109A was expressed in parenchymal cells and not localized exclusively to immune-system cells. Finally, analysis of the putative bovine GPR109A sequence verified that AA residues required for binding niacin in human GPR109A are conserved, suggesting that the bovine sequence identified encodes a functional niacin receptor. The identification of GPR109A in bovine liver, muscle, and brain is a novel finding.

Attenuation of apoptosis in vitro and ischemia/reperfusion injury in vivo in mouse skeletal muscle by P2Y6 receptor activation

Activation of the G(q)-coupled P2Y(6) receptor heterologously expressed in astrocytes significantly attenuates apoptosis induced by tumor necrosis factor alpha (TNFalpha). We have extended the analysis of P2Y(6) receptor-induced cytoprotection to mouse skeletal muscle cells endogenously expressing this receptor. The endogenous P2Y(6) receptor agonist UDP and synthetic agonist MRS2693 protected C2C12 skeletal muscle cells against apoptosis in a concentration-dependent manner (0.1-10 nM) as determined by propidium iodide staining, histochemical analysis using hematoxylin and Hoechst 33258, and DNA fragmentation. The insurmountable P2Y(6) receptor antagonist MRS2578 blocked the protection. TNFalpha-induced apoptosis in C2C12 cells correlated with activation of the transcription factor NF-kappaB. The NF-kappaB activation was attenuated by 10nM MRS2693, which activated the antiapoptic ERK1/2 pathway. In an in vivo mouse hindlimb model, MRS2693 protected against skeletal muscle ischemia/reperfusion injury. The P2Y(6) receptor is a novel cytoprotective receptor that deserves further exploration in ameliorating skeletal muscle injury.

Effects of monensin on metabolic parameters, feeding behavior, and productivity of transition dairy cows

The effects of monensin on transition cow metabolism may be dependent on modulation of feeding behavior, rumen pH, and expression of key metabolic genes. Multiparous Holstein cows were used to determine the effects of monensin (400mg/cow daily) on these variables. Cows were randomly assigned, based on calving date, to control or monensin treatments (n = 16 per treatment) 21 d before their expected calving date, and cows remained on treatments through 21 d postpartum. Feeding behavior and water intake data were collected daily. Liver biopsies were conducted after assessing BCS and BW on d -21, -7, 1, 7, and 21 relative to calving for analysis of triglyceride (TG) content as well as mRNA abundance of cytosolic phosphoenolpyruvate carboxykinase, carnitine palmitoyltransferase 1a, and apolipoprotein B. Blood samples were collected 21, 7, and 4 d before expected calving and 1 (day of calving), 4, 7, 14, and 21 d postpartum for nonesterified fatty acid, β-hydroxybutyrate, glucose, insulin, and haptoglobin analyses. Ruminal pH was collected every 5 min on d 1 through 6 postpartum via a wireless indwelling probe. On d 7 postpartum, a caffeine clearance test was performed to assess liver function. Data were analyzed using mixed models with repeated measures over time. Monensin decreased mean plasma β-hydroxybutyrate (734 vs. 616 ± 41 μM) and peak concentrations (1,076 vs. 777 ± 70 μM on d 4 postpartum). Monensin also decreased time between meals prepartum (143 vs. 126 ± 5.0 min) and postpartum (88.8 vs. 81.4 ± 2.9 min), which was likely related to a smaller ruminal pH standard deviation in the first day after cows changed to a lactation ration (0.31 vs. 0.26 ± 0.015). Monensin also increased liver mRNA abundance of carnitine palmitoyltransferase 1a (0.10 vs. 0.15 ± 0.002 arbitrary units), which corresponded to a slower rate of liver TG accumulation from d -7 to +7 (412 vs. 128 ± 83 mg of TG/g of protein over this time period). No significant effects of monensin supplementation were observed on milk production, liver cytosolic phosphoenolpyruvate carboxykinase, apolipoprotein B, plasma nonesterified fatty acid, glucose, insulin, or haptoglobin. No effects on disease incidence were detected, but sample size was small for detecting such effects. Overall, results confirm that the effects of monensin on transition cows extend beyond altered propionate flux.

Mechanism of glycogen supercompensation in rat skeletal muscle cultures

A model to study glycogen supercompensation (the significant increase in glycogen content above basal level) in primary rat skeletal muscle culture was established. Glycogen was completely depleted in differentiated myotubes by 2 h of electrical stimulation or exposure to hypoxia during incubation in medium devoid of glucose. Thereafter, cells were incubated in medium containing glucose, and glycogen supercompensation was clearly observed in treated myotubes after 72 h. Peak glycogen levels were obtained after 120 h, averaging 2.5 and 4 fold above control values in the stimulated- and hypoxia-treated cells, respectively. Glycogen synthase activity increased and phosphorylase activity decreased continuously during 120 h of recovery in the treated cells. Rates of 2-deoxyglucose uptake were significantly elevated in the treated cells at 96 and 120 h, averaging 1.4–2 fold above control values. Glycogenin content increased slightly in the treated cells after 48 h (1.2 fold vs. control) and then increased considerably, achieving peak values after 120 h (2 fold vs. control). The results demonstrate two phases of glycogen supercompensation: the first phase depends primarily on activation of glycogen synthase and inactivation of phosphorylase; the second phase includes increases in glucose uptake and glycogenin level.

Enhanced connexin-43 and alpha-sarcomeric actin expression in cultured heart myocytes exposed to triiodo-L-thyronine.

This study examined whether triiodo-l-thyronine (T3) affects the expression of the major intercellular channel protein, connexin-43, and contractile protein α-sarcomeric actin. Cultured cardiomyocytes from newborn rats were treated on day three in culture with 10 or 100 nM T3 and examined 48 and 72 h thereafter. Treated and untreated cells were examined by immunofluorescence and electron microscopy. Expression levels of Cx43 and sarcomeric α-actin were monitored by Western blot analysis. Immunofluorescence labeling showed cell membrane location of Cx43 in punctuate gap junctions, whereby fluorescence signal area was significantly higher in cultured cardiomyocytes exposed to T3. This correlated with electron microscopical findings showing increased numbers and size of gap junction profiles, as well as with a significant dose-dependent increase of Cx43 expression detected by Western blot. Immunofluorescence of sarcomeric α-actin was enhanced and its expression increased dose- and time-dependently in T3-treated cultured heart myocytes. However, exposure to the higher dosage (100 nM) of T3 caused mild disintegration of sarcomeric α-actin in some myocytes, suggesting an over-dosage. The results indicate that T3 up-regulates Cx43 and accelerates gap junction formation in cultured neonatal cardiomyocytes. They suggest that thyroid status cannot only modulate the mechanical function of cardiomyocytes but also cell-to-cell communication essential for myocardial electrical and metabolic synchronizations.

Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.

Activation of Adenosine A1 and A3 Receptors Protects Mitochondria during Hypoxia in Cardiomyocytes by Distinct Mechanisms

Results of many investigations indicate that activation of adenosine (ADO) A1 and A3 receptors (A1Rs and A3Rs) elicits delayed protection against infarction, ischemia or hypoxia and that both A1R and A3R induce cardioprotection through opening of KATP channels. We suppose that opening of KATP channels may not be the only final mediator of cardioprotection. The protection of the mitochondrial respiratory chain and its impact on mitochondrial bioenergetics after ADO receptor activation may be achieved by different mechanisms. The contribution of mitochondrial and sarcolemmal KATP channels, the rate of mitochondrial ATP synthesis and redox state of mitochondria were compared in normoxic and hypoxic conditions on cultured newborn cardiomyocytes. Activation of both subtypes of ADO receptors induces certain decrease in energy supply and simultaneously promotes preservation of adequate amounts of ATP and maintenance of mitochondrial metabolism on a level sufficient for cell survival. It was found that neither diazoxide nor A1R agonist CCPA nor A3R agonist Cl-IB-MECA modified mitochondrial membrane potential in intact cells. Activation of adenosine A1 receptor slowed down the ΔΨ repolarization. Diazoxide also decreases the rate of energization capacity in living cardiomyocytes upon succinate oxidation. The A3R agonist Cl-IB-MECA did not affect mitochondrial bioenergetics in normoxic cardiomyocytes. It was shown that A3 adenosine receptor stimulation modulates the sarcoplasmic reticulum (SR) Ca2+ channel and may regulate Ca2+ overloading. In conclusion, our data establish that adenosine can mediate myocardial protection by acting on A1 and A3 adenosine receptors. However, the cascades of events involved in cardioprotection against hypoxia appear to be distinct for A1 and A3 receptor signaling.