Effects of IGF-I, IGF-II, bFGF and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts.

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth factors and the recruitment of free ribosomes to polysomes was quantified. All experiments were conducted on C2C12 myoblasts (24 h prior to induction of fusion) and differentiating myoblasts (24 h after induction of fusion). After the 2 h incubation, cells were rinsed with phosphate buffered saline and quickly frozen at -80 degrees C. Cell lysates were fractionated on 15-60% sucrose gradients by centrifugation at 200,000 x g for 1 h. Absorbance at 254 nm was recorded continuously across the gradient. The response to each of the four growth factors, IGF-I and-II, basic fibroblast growth factor (FGF), and platelet-derived growth factor was a decrease (P < 0.05) in monosome peak height and a increase (P < 0.05) in polysome percentage (P < 0.05). All responses were linear, except IGF-I, and the monosome peak height response to FGF which were quadratic (P < 0.05). None of the growth factors had a significant effect (P > 0.05) on RNA concentrations over the 2-h incubation. Protein content did not vary due to growth factor or level of treatment. This corroborates the hypothesis that the acute increase of protein synthesis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesis. These results suggest that we can study the mechanisms regulating protein synthesis in muscle cells effectively by studying shifts in ribosomal activity. This method gave more consistent results than the H3-tyrosine incorporation and has the added benefit of not requiring the use of radioactivity. The strong correlation between monosome peak heights and percentage polysomes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysomes for other uses. The similarity of response among the various growth factors may indicate a common mechanism for increasing the initiation of protein synthesis.     

Protein kinase C modulates insulin action in human skeletal muscle

There is good evidence from cell lines and rodents that elevated protein kinase C (PKC) overexpression/activity causes insulin resistance. Therefore, the present study determined the effects of PKC activation/inhibition on insulin-mediated glucose transport in incubated human skeletal muscle and primary adipocytes to discern a potential role for PKC in insulin action. Rectus abdominus muscle strips or adipocytes from obese, insulin-resistant, and insulin-sensitive patients were incubated in vitro under basal and insulin (100 nM)-stimulated conditions in the presence of GF 109203X (GF), a PKC inhibitor, or 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a PKC activator. PKC inhibition had no effect on basal glucose transport. GF increased (P < 0.05) insulin-stimulated 2-deoxyglucose (2-DOG) transport approximately twofold above basal. GF plus insulin also increased (P < 0.05) insulin receptor tyrosine phosphorylation 48% and phosphatidylinositol 3-kinase (PI 3-kinase) activity approximately 50% (P < 0.05) vs. insulin treatment alone. Similar results for GF on glucose uptake were observed in human primary adipocytes. Further support for the hypothesis that elevated PKC activity is related to insulin resistance comes from the finding that PKC activation by dPPA was associated with a 40% decrease (P < 0.05) in insulin-stimulated 2-DOG transport. Incubation of insulin-sensitive muscles with GF also resulted in enhanced insulin action ( approximately 3-fold above basal). These data demonstrate that certain PKC inhibitors augment insulin-mediated glucose uptake and suggest that PKC may modulate insulin action in human skeletal muscle.     

Tyrosine kinase and protein kinase C regulate L-type Ca(2+) current cooperatively in human atrial myocytes

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.     

Influence of caffeine on metabolic responses of men at rest in 28 and 5 degrees C

Cold stress and caffeine ingestion are each reported to increase plasma catecholamines, free fatty acid (FFA) concentrations, and energy metabolism. This study examined the possible interaction of these two metabolic challenges in four double-blind counterbalanced trials. Young adult men (n = 6) ingested caffeine (5 mg/kg) or placebo (dextrose, 5 mg/kg) and rested for 2 h in 28 or 5 degrees C air. Cold stress alone elevated (P less than 0.05) plasma norepinephrine, metabolism (O2 consumption, VO2), and respiratory exchange ratio (RER). Caffeine alone increased (P less than 0.05) plasma epinephrine and FFA but not RER. When the two challenges were combined (caffeine plus 5 degrees C for 2 h) norepinephrine and epinephrine were increased (P less than 0.05) as was FFA. However, VO2, RER, and skin and rectal temperatures were not different from the responses observed at 5 degrees C after placebo ingestion. The data suggest that caffeine selectively increases plasma epinephrine, whereas cold air increases norepinephrine. During the cold exposure, increasing epinephrine and FFA above normal levels did not appear to influence the metabolic or thermal responses to the cold stress. In fact the increase in RER suggested a greater carbohydrate oxidation.     

Gestational and hormonal regulation of human placental lipoprotein lipase

The fetal demand for FFA increases as gestation proceeds, and LPL represents one potential mechanism for increasing placental lipid transport. We examined LPL activity and protein expression in first trimester and term human placenta. The LPL activity was 3-fold higher in term (n = 7; P < 0.05) compared with first trimester (n = 6) placentas. The LPL expression appeared lower in microvillous membrane from first trimester (n = 2) compared with term (n = 2) placentas. We incubated isolated placental villous fragments with a variety of effectors [GW 1929, estradiol, insulin, cortisol, epinephrine, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha] for 1, 3, and 24 h to investigate potential regulatory mechanisms. Decreased LPL activity was observed after 24 h of incubation with estradiol (1 micro g/ml), insulin, cortisol, and IGF-1 (n = 12; P < 0.05). We observed an increase in LPL activity after 3 h of incubation with estradiol (20 ng/ml) or hyperglycemic medium plus insulin (n = 7; P < 0.05). To conclude, we suggest that the gestational increase in placental LPL activity represents an important mechanism to enhance placental FFA transport in late pregnancy. Hormonal regulation of placental LPL activity by insulin, cortisol, IGF-1, and estradiol may be involved in gestational changes and in alterations in LPL activity in pregnancies complicated by altered fetal growth.     

Beta-adrenergic agonist hyperplastic effect is associated with increased fibronectin gene expression and not mitogen-activated protein kinase modulation in C2C12 cells

Beta-adrenergic agonists (beta-AA) enhance protein accretion in skeletal muscles. This stimulation is characterized by increased protein synthesis, increased expression of myofibrillar protein genes and a depression in protein degradation in animals, and increased proliferation and DNA synthesis in muscle cells in vitro. The mechanism or signal path in muscle whereby beta-AA would elicit these physiological effects upon binding to the G protein-coupled beta-adrenergic receptor (beta-AR) is unclear. C2C12 myoblasts were used to determine beta-AR ligand binding characteristics, cyclic AMP synthesis in response to isoproterenol (ISO) stimulation, and effects of ISO on DNA synthesis, mitogen activated protein kinase (MAPK), and fibronectin (FN) gene expression. Results showed that C2C12 cells possess beta-AR which are specific, saturable, and of high affinity (Kd = 0.2 nM). Forskolin and ISO stimulated cAMP production by = 20-fold (P<0.001) and 17-fold (P<0.001), respectively. ISO and the cAMP analog, 8-bromo-cAMP (8-BC) stimulated DNA synthesis in proliferating cells by 150% (P<0.05) and 200% (P<0.01), respectively, without modulating MAPK activity, whereas addition of fetal bovine serum to culture resulted in a 500% increase (P<0.01) in DNA synthesis and MAPK activation. DNA synthesis in C2C12 cells treated with ISO, 8-BC, or FBS was abolished in the presence of 25 microM PD098059, an MAPK-kinase inhibitor, suggesting that an MAPK-dependent pathway is likely involved in C2C12 proliferation. During cAMP elevating agent stimulation, basal MAPK activity may be sufficient, in the presence of other putative signaling molecules, to support proliferation in these cells. ISO or 8-BC treatment increased FN mRNA by three- and seven-fold, respectively, in growing C2C12 cells implying a connection between increased DNA synthesis and FN gene expression.     

Vitamin C, resveratrol and lipoic acid actions on isolated rat liver mitochondria: all antioxidants but different

Modulating mitochondrial antioxidant status is a nutritional issue of great interest in the treatment or prevention of several oxidative stress related diseases such as obesity. Thus, the aim of the present study was to analyze the effects of three antioxidants on hepatic mitochondrial function and antioxidant status. Isolated rat liver mitochondria were incubated with vitamin C, resveratrol and lipoic acid. The activity of antioxidant enzymes (manganese superoxide dismutase and glutathione peroxidase), ROS generation and respiratory parameters (RCR, P/O ratio and respiratory states) were measured. Vitamin C influenced mitochondrial function by decreasing of ROS generation (P < 0.0001), by stimulating the activity of manganese superoxide dismutase (197.60 ± 35.99%; P < 0.001) as well as glutathione peroxidase (15.70 ± 5.76%; P < 0.05) and by altering the activity of the electron transport chain, mainly by decreasing the P/O ratio (P < 0.05). Resveratrol induced a significant increase in manganese superoxide dismutase activity (160 ± 11.78%; P < 0.0001) and a decrease in ROS generation (P < 0.05 to P < 0.0001). By contrast, lipoic acid inhibited glutathione peroxidase activity (16.48 ± 3.27%; P < 0.05) and induced the uncoupling of the electron transport chain (P < 0.01). Moreover, this antioxidant induced a strong decrease in the P/O ratio (P < 0.05 to P < 0.0001). In conclusion, our results suggest that the three tested antioxidants produced direct effects on mitochondrial function, although the magnitude and intensity of these actions were significantly different, which may have implications when administrated as antioxidants.     

Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo

Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.     

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.     

Properties of epinephrine-induced activation of cardiac adenosine 3′,5′-monophosphate-dependent protein kinase

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (—cyclic AMP/+cyclic AMP) of 12 000 × g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 μg/kg) resulted from an increase in independent protein kinase activity (—cyclic 2 AMP) without a change in total protein observ activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 μg/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

Ethanol ingestion via glutathione depletion impairs alveolar epithelial barrier function in rats

We determined that rats fed a liquid diet containing ethanol (36% of calories) for 6 wk had decreased (P < 0.05) net vectorial fluid transport and increased (P < 0.05) bidirectional protein permeability across the alveolar epithelium in vivo compared with rats fed a control diet. However, both groups increased (P < 0.05) fluid transport in response to epinephrine (10(-5) M) stimulation, indicating that transcellular sodium transport was intact. In parallel, type II cells isolated from ethanol-fed rats and cultured for 8 days formed a more permeable monolayer as reflected by increased (P < 0.05) leak of [(14)C]inulin. However, type II cells from ethanol-fed rats had more sodium-permeant channels in their apical membranes than type II cells isolated from control-fed rats, consistent with the preserved response to epinephrine in vivo. Finally, the alveolar epithelium of ethanol-fed rats supplemented with L-2-oxothiaxolidine-4-carboxylate (Procysteine), a glutathione precursor, had the same (P < 0.05) net vectorial fluid transport and bidirectional protein permeability in vivo and permeability to [(14)C]inulin in vitro as control-fed rats. We conclude that chronic ethanol ingestion via glutathione deficiency increases alveolar epithelial intercellular permeability and, despite preserved or even enhanced transcellular sodium transport, renders the alveolar epithelium susceptible to acute edematous injury.     

In vivo and in vitro iron deficiency reduces protein kinase C activity and translocation in murine splenic and purified T cells.

We investigated the effects of iron deficiency anemia, iron repletion, and iron chelation by deferoxamine on protein kinase C (PKC) activity, an enzyme that plays a crucial role on T lymphocyte proliferation. The study involved 23 control (C), 18 pairfed (PF), and 24 iron deficient (ID) mice or ID mice that were repleted for 3 (n = 14), 7 (n = 17), or 14 (n = 14) days. The low iron (0.09 mmol iron/kg) and iron-supplemented (0.9 mmol iron/kg) diets were fed to mice for 53 days. Mean hemoglobin, hematocrit, and liver iron stores of ID mice were one third of those of C mice. Lymphocyte proliferation was reduced (P < 0.05) in spleen and purified T cells in ID but not PF mice. In concanavalin A, phytohemagglutinin, and anti-CD3 antibody-treated and untreated cells that were incubated in serum-free and serum-containing medium, PKC activity was significantly (P < 0.05) reduced in ID but not PF mice and returned to normal before correction of anemia. In mitogen-treated cells, while the ratios of membrane-bound to cytosol activity increased nearly seven-fold (from 0.4-0.63 in resting cells to 1.43-7.23) in spleen cells from C, PF, and repleted mice and 11-fold in T cells (P < 0.005), they remained below 1 in ID mice suggesting reduced translocation. In vitro iron chelation by deferoxamine for 120 min prior to cell activation reduced (P < 0.05) PKC activity by 46-60% in C and PF and 28-53% in ID mice. The data suggest that: 1) it is iron-deficiency but not anemia or differences in the proportion of immunocompetent T cells that reduced PKC activity in cells from ID mice; 2) reduced PKC translocation may play an important role on altered lymphocyte proliferation and associated functions in iron-deficient individuals.     

Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers

Effects of inhaled nitric oxide (NO) on human platelet function are controversial. It is uncertain whether intraplatelet cGMP mediates the effect of inhaled NO on platelet function. We investigated the effect of 30 ppm inhaled NO on platelet aggregation and plasma and intraplatelet cGMP in 12 subjects. We performed platelet aggregation studies by using a photooptical aggregometer and five agonists (ADP, collagen, epinephrine, arachidonic acid, and ristocetin). During inhalation, the maximal extent of platelet aggregation decreased by 75% with epinephrine (P < 0.005), 56% with collagen (P < 0.005), and 20% with arachidonic acid (P < 0.05). Responses to ADP (8% P > 0.05) and ristocetin (5% P > 0.05) were unaffected. Platelet aggregation velocity decreased by 64% with collagen (P < 0.005), 60% with epinephrine (P < 0.05), 33% with arachidonic acid (P < 0.05), and 14% with ADP (P > 0.05). Plasma cGMP levels increased from 2.58 +/- 0.43 to 9.99 +/- 5.57 pmol/ml (P < 0.005), intraplatelet cGMP levels were unchanged (means +/- SD: 1.96 +/- 0.58 vs. 2.71 +/- 1.67 pmol/109 platelets; P > 0.05). Inhaled NO inhibits platelet aggregation via a cGMP independent mechanism.     

Altered N,P and C dynamics with absence of fire in Eucalyptus forests affected by premature decline

Absence of fire is increasingly recognized as an important driver of soil nutrient budgets in Eucalyptus forest, especially in forests affected by premature Eucalyptus decline, due to the effects of soil nutrient accumulation on nutrient balances and forest community dynamics. In this study, we present a dataset of soil and foliar nutrient analyses, and vegetation measurements from a fire chronosequence survey in native E. delegatensis forest. Measured indices include total soil and extractable soil nitrogen (N), or phosphorus (P), soil organic carbon (C), soil acid‐phosphatase (PME) activity, foliar N and foliar P, and understorey and overstorey vegetation canopy height. We show that in some cases indices are strongly linked to time since fire (2–46 years). Time since fire correlated positively with foliar N, total and extractable soil N, soil organic C, and also soil PME activity; the latter an indicator of biotic P demand. Differences in the strength of these relationships were apparent between two geology types, with stronger relationships on the potentially less‐fertile geology. The strong positive correlation with time since fire and understorey canopy height reflected increasing shrub biomass and thickening of the shrub layer. The strong positive correlation for soil or foliar N, but not P, with time since fire, indicates that P does not increase relative to N over time. P may, therefore, become limiting to growth in this plant community. Similarly, the significantly higher concentrations of soil N but not P, also found in both older and long‐unburnt forest stands (>100 years since management), may exacerbate a situation of soil nutrient limitation over several decades. A characteristic feature of long unmanaged stands is a developing tea tree (Leptospermum sp.) understorey, which may benefit from elevated soil N availability and increasing organic C accumulation with prolonged fire absence. This increased shrub biomass would outcompete Eucalyptus for resources, including soil nutrients and water.     

Effects of plasma epinephrine on fat metabolism during exercise: interactions with exercise intensity

This study determined the effects of elevated plasma epinephrine on fat metabolism during exercise. On four occasions, seven moderately trained subjects cycled at 25% of peak oxygen consumption (VO(2 peak)) for 60 min. After 15 min of exercise, subjects were intravenously infused with low (0.96 +/- 0.10 nM), moderate (1.92 +/- 0.24 nM), or high (3.44 +/- 0.50 nM) levels (all P < 0.05) of epinephrine to increase plasma epinephrine above control (Con; 0.59 +/- 0.10 nM). During the interval between 35 and 55 min of exercise, lipolysis [i.e., rate of appearance of glycerol] increased above Con (4.9 +/- 0.5 micromol. kg(-1). min(-1)) with low, moderate, and high (6.5 +/- 0.5, 7.1 +/- 0.8, and 10.6 +/- 1.2 micromol. kg(-1). min(-1), respectively; all P < 0.05) levels of epinephrine despite simultaneous increases in plasma insulin. The release of fatty acid into plasma also increased progressively with the graded epinephrine infusions. However, fatty acid oxidation was lower than Con (11.1 +/- 0.8 micromol. kg(-1). min(-1)) during moderate and high levels (8.7 +/- 0.7 and 8.1 +/- 0.9 micromol. kg(-1). min(-1), respectively; P < 0.05). In one additional trial, the same subjects exercised at 45% VO(2 peak) without epinephrine infusion, which produced a plasma epinephrine concentration identical to low levels. However, lipolysis was lower (i.e., 5.5 +/- 0.6 vs. 6.5 +/- 0.5 micromol. kg(-1). min(-1); P < 0.05). In conclusion, elevations in plasma epinephrine concentration during exercise at 25% of VO(2 peak) progressively increase whole body lipolysis but decrease fatty acid oxidation. Last, increasing exercise intensity from 25 to 45% VO(2 peak) attenuates the lipolytic actions of epinephrine.