Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet

We have shown that first-pass intestinal metabolism is necessary for approximately 50% of whole body arginine synthesis from its major precursor proline in neonatal piglets. Furthermore, the intestine is not the site of increased arginine synthesis observed during dietary arginine deficiency. Primed constant intravenous (iv) and intraportal (ip) infusions of L-[U-14C]proline, and iv infusion of either L-[guanido-14C]arginine or L-[4,5-3H]arginine were used to measure first-pass hepatic arginine synthesis in piglets enterally fed either deficient (0.20 g.kg(-1).day(-1)) or generous (1.80 g.kg(-1).day(-1)) quantities of arginine for 5 days. Conversion of arginine to other urea cycle intermediates and arginine recycling were also calculated for both dietary treatments. Arginine synthesis (g.kg(-1).day(-1)) from proline was greater in piglets (P < 0.05) fed the deficient arginine diet in both the presence (generous: 0.07; deficient: 0.17; pooled SE = 0.01) and absence (generous: 0.06; deficient: 0.20; pooled SE = 0.01) of first-pass hepatic metabolism. There was no net arginine synthesis from proline during first-pass hepatic metabolism regardless of arginine intake. Arginine conversion to urea, citrulline, and ornithine was significantly greater (P < 0.05) in piglets fed the generous arginine diet. Calculated arginine fluxes were significantly lower (P = 0.01) for [4,5-3H]arginine than for [guanido-14C]arginine, and the discrepancy between the values was greater in piglets fed the deficient arginine diet (35% vs. 20%). Collectively, these findings show that first-pass hepatic metabolism is not a site of net arginine synthesis and that piglets conserve dietary arginine in times of deficiency by decreasing hydrolysis and increasing recycling.     

Hypothalamic protein profiles associated with inhibited feed intake of ducks fed with insufficient dietary arginine

An experiment was conducted to investigate the effect of arginine on feed intake regulation. One hundred and twenty six 1-day-old male White Pekin ducks (Anas platyrhynchos domestica) were randomly were allotted to one of two dietary treatments. The birds were fed diets containing 0.71% (deficient) or 1.27% (sufficient) arginine for 3 weeks. At 21 days of age, feed intake was determined and hypothalamic protein profiles were analyzed using isobaric tags for relative and absolute quantification technique. The birds fed with arginine-deficient diet had a lower final live BW and cumulative feed intake (P<0.01) than those fed with arginine-sufficient diet. A total of 16 proteins were identified in the hypothalamus with >1.5-fold expressional changes between arginine-deficient and -sufficient dietary treatments. Nine of these proteins were upregulated and seven of them were downregulated. The identified proteins could be regrouped into six categories: protein processing, carbohydrate metabolism and energy production, transporter, cytoskeleton, immunity and neuronal development. Dietary arginine deficiency decreased expression of proteins involved in energy production (glycine amidinotransferase, aldolase B fructose-bisphosphate, aconitase, transaldolase, 6-phosphofructokinase type C-like) and oxygen transportation (haemoglobin subunit α expression). The proteomic alterations described here provides valuable insights into the interactions of arginine with appetite.     

Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.     

Qualitative and quantitative comparison of gut bacterial colonization in enterally and parenterally fed neonatal pigs

Total parenteral nutrition (TPN) has been associated with mucosal atrophy, impaired gut barrier function, and translocation of luminal bacteria with resultant sepsis in preterm human infants. Currently, we examined the effects of enteral (ENT) or TPN treatments on translocation events in neonatal pigs and on colonization and composition of microbiota in the neonatal gut. Newborn, colostrum-deprived pigs (<24 hours old) were fitted with intravenous catheters and were fed either ENT (n = 13) or TPN (n = 13) for 7 days. After 7 days of treatment, pigs were euthanized and samples were collected for bacterial culture from the blood, intestinal tract and organs. ENT pigs had increased numbers of bacterial genera isolated, higher concentrations of bacteria (CFU/g), and increased colonization of all segments of the intestinal tract compared to the TPN pigs. Translocation of bacteria from the intestinal tract to tissues or blood was similar (8 of 13) for both groups. The ENT group had 1/13 positive for Clostridium difficile toxin A whereas the TPN group had 5/13. We concluded that ENT favored increased bacterial concentrations comprised of more speciation in the gastrointestinal tract compared to TPN, and that TPN-treated piglets were at higher risk of colonization by toxin-expressing strains of C. difficile.     

Arginine synthesis from enteral glutamine in healthy adults in the fed state

Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.     

Myostatin expression is regulated by underfeeding and neonatal programming in rats

Confusing results have been reported regarding the influence of nutritional status on myostatin levels. Some studies indicate that short-term fasting results in increased myostatin mRNA levels in skeletal muscle, evident in several species. In contrast, other studies have demonstrated either a decrease or no change in myostatin levels during fasting. In the present study, we investigated the effect of different patterns of food deprivation on muscle myostatin expression in both newborn and adult rats. Adjustment of litter size in neonatal rats is a well-established model to study the effect of early overfeeding or underfeeding on body composition and in this study resulted in modifications in the pattern of muscle myostatin expression. Rat pups growing in large litters (22–24 newborns) showed a decrease in muscle myostatin mRNA and protein levels at 24 days of age. Interestingly, these effects were maintained at 60 days of age despite rats having free access to food since weaning, thus suggesting that changes in myostatin expression induced by neonatal reduction of food intake are long-lasting. In contrast, no changes in myostatin mRNA levels were observed in adult rats when food intake was decreased during 7 days by either food restriction or central leptin treatment. Similar results were obtained when food restriction was maintained in adult rats for a longer period (7 weeks), despite significant muscle loss. Overall, these data suggest that myostatin gene expression is programmed by nutritional status in neonatal life.     

A multitracer stable isotope quantification of the effects of arginine intake on whole body arginine metabolism in neonatal piglets

We have previously shown that deficient arginine intake increased the rate of endogenous arginine synthesis from proline. In this paper, we report in vivo quantification of the effects of arginine intake on total endogenous arginine synthesis, on the rates of conversion between arginine, citrulline, ornithine, and proline, and on nitric oxide synthesis. Male piglets, with gastric catheters for diet and isotope infusion and femoral vein catheters for blood sampling, received a complete diet for 2 days and then either a generous (+Arg; 1.80 g x kg(-1) x day(-1); n = 5) or deficient (-Arg; 0.20 g.kg(-1).day(-1); n = 5) arginine diet for 5 days. On day 7, piglets received a primed, constant infusion of [guanido-(15)N(2)]arginine, [ureido-(13)C;5,5-(2)H(2)]citrulline, [U-(13)C(5)]ornithine, and [(15)N;U-(13)C(5)]proline in an integrated study of the metabolism of arginine and its precursors. Arginine synthesis (micromol x kg(-1) x h(-1)) from both proline (+Arg: 42, -Arg: 74, pooled SE: 5) and citrulline (+Arg: 67, -Arg: 120; pooled SE: 15) were higher in piglets receiving the -Arg diet (P < 0.05); and for both diets proline accounted for approximately 60% of total endogenous arginine synthesis. The conversion of proline to citrulline (+Arg: 39, -Arg: 67, pooled SE: 6) was similar to the proline-to-arginine conversion, confirming that citrulline formation limits arginine synthesis from proline in piglets. Nitric oxide synthesis (micromol x kg(-1) x h(-1)), measured by the rate conversion of [guanido-(15)N(2)]arginine to [ureido-(15)N]citrulline, was greater in piglets receiving the +Arg diet (105) than in those receiving the -Arg diet (46, pooled SE: 10; P < 0.05). This multi-isotope method successfully allowed many aspects of arginine metabolism to be quantified simultaneously in vivo.     

Lipid metabolism in pigs fed supplemental conjugated linoleic acid and/or dietary arginine

We proposed that the combination of conjugated linoleic acid (CLA) and arginine would decrease adiposity by depressing lipid synthesis in liver and adipose tissues of growing pigs. Pigs were allotted to treatments in a 2 × 2 factorial design with two lipids (CLA or canola oil) and two amino acids [l-arginine or l-alanine (isonitrogenous control)]; supplements were provided from 80 to 110 kg body weight (approximately 4 weeks). Treatment groups (n = 4) were: control (2.05% l-alanine plus 1% canola oil); CLA (2.05% l-alanine plus 1% CLA); arginine (1.0% l-arginine plus 1.0% canola oil); arginine plus CLA (1.0% arginine plus 1.0% CLA). Arginine increased backfat thickness (P = 0.07) in the absence or presence of CLA, and arginine supplementation increased subcutaneous and retroperitoneal adipocyte volume, especially in combination with dietary CLA (interaction P = 0.001). Arginine increased palmitate incorporation into total lipids by over 60% in liver (P = 0.07). Dietary CLA increased palmitate incorporation into lipids in longissimus muscle by over 100% (P = 0.01), and CLA increased longissimus muscle lipid by nearly 20%. CLA increased glucose oxidation to CO₂ by over 80% in retroperitoneal and subcutaneous adipose tissues (P = 0.04), and doubled palmitate oxidation to CO₂ in intestinal duodenal mucosal cells (P = 0.07). Arginine supplementation decreased muscle pH at 45 min postmortem (P = 0.001), indicating elevated early postmortem glycolysis, and CLA and arginine independently increased PGC-1α gene expression in longissimus muscle. CLA but not arginine depressed mTOR gene expression in intestinal duodenal mucosal cells. CLA decreased serum insulin by 50% (P = 0.02) but increased serum triacylglycerols by over 40%. CLA supplementation increased (P ≤ 0.01) total saturated fatty acids in liver and adipose tissue. In conclusion, neither CLA nor arginine depressed tissue lipid synthesis in growing/finishing pigs, and in fact dietary CLA promoted elevated intramuscular lipid and arginine increased carcass adiposity.     

Beating rate of isolated neonatal cardiomyocytes is regulated by the stable microtubule subset

We investigated the roles of microtubule (MT) dynamics (growth and shrinkage), the stable, nongrowing MT subset, the posttranslationally detyrosinated MT subset, and artificially elevated tubulin levels in the negative regulation of heart cell beating rate. We manipulated the MT populations in isolated, neonatal cardiomyocytes obtained from normal animals in several ways and then measured heart cell beating rate directly. We found that the stabilized population of MTs was sufficient to maintain a normal beating rate, whereas MT dynamics and detyrosination made no observable contribution. Furthermore, by directly and acutely increasing the level of tubulin within otherwise normally beating cells, we found that the increased tubulin (and MT) levels further depressed the beating rate. In conclusion, the stabilized MT subset is sufficient to maintain the normal beating rate in these cells, whereas increasing the MT density depresses it.     

Identification of novel protein targets regulated by maternal dietary fatty acid composition in neonatal rat liver

Polyunsaturated fatty acids regulate metabolic pathways, which in early development could have important consequences to adaptation to extra-uterine life and programming of metabolic pathways. Female rats were fed one of two diets identical in all nutrients, except that the fat in one diet was high in unsaturated fatty acids (UFA) and the other low UFA, through gestation and lactation. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis of protein extracts from 3-day old pup liver resolved over 800 proteins. Employing MALDI-TOF MS and peptide mapping we identified 11 proteins that differed more than three-fold between the groups, 10 up regulated and one down regulated in the high UFA group. The up-regulated proteins included fructose-1,6-bisphosphatase 1, glycerol-3-phosphate dehydrogenase, galactokinase 1, 40S ribosomal protein SA, elongation factor 1-gamma, protein disulfide-isomerase A6, catalase, cytokeratin-8 and 60 kDa heat shock protein, and the down-regulated protein was argininosuccinate synthase, none having been previously reported to be regulated by fatty acids in the developing liver. We further determined that fructose-1,6-biphosphatase is acetylated at the N-terminus. We demonstrate that early fatty acid nutrition impacts hepatic metabolic pathways relevant to gluconeogenesis, redox balance and nitric oxide signaling.     

Identification of novel protein targets regulated by maternal dietary fatty acid composition in neonatal rat liver

Polyunsaturated fatty acids regulate metabolic pathways, which in early development could have important consequences to adaptation to extra-uterine life and programming of metabolic pathways. Female rats were fed one of two diets identical in all nutrients, except that the fat in one diet was high in unsaturated fatty acids (UFA) and the other low UFA, through gestation and lactation. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis of protein extracts from 3-day old pup liver resolved over 800 proteins. Employing MALDI-TOF MS and peptide mapping we identified 11 proteins that differed more than three-fold between the groups, 10 up regulated and one down regulated in the high UFA group. The up-regulated proteins included fructose-1,6-bisphosphatase 1, glycerol-3-phosphate dehydrogenase, galactokinase 1, 40S ribosomal protein SA, elongation factor 1-gamma, protein disulfide-isomerase A6, catalase, cytokeratin-8 and 60 kDa heat shock protein, and the down-regulated protein was argininosuccinate synthase, none having been previously reported to be regulated by fatty acids in the developing liver. We further determined that fructose-1,6-biphosphatase is acetylated at the N-terminus. We demonstrate that early fatty acid nutrition impacts hepatic metabolic pathways relevant to gluconeogenesis, redox balance and nitric oxide signaling.     

HGF synthesis in human lung fibroblasts is regulated by oncostatin M

Oncostatin M (OSM) is a IL-6 family cytokine locally produced in acute lung injury. Its profibrotic properties suggest a role in lung wound repair. Hepatocyte growth factor (HGF), produced by fibroblasts, is involved in pulmonary epithelial repair. We investigated the role of OSM in HGF synthesis by human lung fibroblasts. We showed that OSM upregulated HGF mRNA in MRC5 cells and in human lung fibroblasts, whereas IL-6 and leukemia inhibitory factor did not. OSM induced HGF secretion to a similar extent as IL-1beta in both a time- and dose-dependent manner. HGF was released in its cleaved mature form, and its secretion was completely inhibited in the presence of cycloheximide, indicating a de novo protein synthesis. OSM in combination with prostaglandin E(2), a powerful HGF inductor, led to an additive effect. OSM and indomethacin in combination further increased HGF secretion. This could be explained, at least in part, by a moderate upregulation of specific OSM receptor beta mRNA expression through cyclooxygenase inhibition. These results demonstrate that OSM-induced HGF synthesis did not involve a PGE(2) pathway. OSM-induced HGF secretion was inhibited by PD-98059 (a specific pharmacological inhibitor of ERK1/2), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor) by 70, 82, and 100%, respectively, whereas basal HGF secretion was only inhibited by SP-600125 by 30%. Our results demonstrate a specific upregulation of HGF synthesis by OSM, most likely through a MAPK pathway, and support the suggestion that OSM may participate in lung repair through HGF production.     

TMV protein synthesis is not translationally regulated by heat shock

Summary Tobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.     

Lung injury in the neonatal piglet caused by hyperoxia and mechanical ventilation

Neonatal lung injury from hyperoxia and mechanical hyperventilation was studied in newborn piglets hyperventilated (arterial PCO2 15-20 Torr) for 24-48 h with 100% O2 and compared with unventilated controls. Pulmonary function testing was performed, and biochemical indicators of lung injury were analyzed from tracheobronchial aspirates at 0, 24, and 48 h. Lung sections were obtained for light and electron microscopy, and bronchoalveolar lavage fluid was analyzed for surfactant composition and activity. At 24 h significant changes in tracheobronchial aspirate albumin concentrations (up 78%) and percent of polymorphonuclear cells (up 16%) were demonstrated. At 48 h a 35% decrease in dynamic lung compliance (P less than 0.05) and a 36% increase in pulmonary resistance (P less than 0.05) were noted. Further biochemical abnormalities occurred with total cell counts increased by 271% (P less than 0.02), albumin 163% (P less than 0.05), total protein 217% (P less than 0.01), and elastase 108% (P less than 0.02). Pathological analyses revealed mild lung injury at 24 h and marked inflammation, abnormal inflation patterns, flattening of Clara cells, fibrinous exudate and edema, early collagen formation, and cell necrosis observed at 48 h. Bronchoalveolar lavage surfactant had normal biophysical activity. Results demonstrate that exposure of neonatal piglets to O2 and mechanical hyperventilation for 48 h cause severe progressive lung injury.