Mary A. Dudley

Parenteral nutrition selectively decreases protein synthesis in the small intestine

We investigated the effects of an elemental diet fed parenterally or enterally on total mucosal protein and lactase phlorizin hydrolase (LPH) synthesis. Catheters were placed in the stomach, jugular vein, and carotid artery of 12 3-day-old pigs. Half of the animals were given an elemental regimen enterally and the other half parenterally. Six days later, animals were infused intravenously with [2H3]leucine for 6 h and killed, and the midjejunum of each animal was collected for analysis. The weight of the midjejunum was 8 +/- 1.5 and 17 +/- 1.6 g in parenterally fed and enterally fed piglets, respectively. LPH activities (mumol.min-1.g protein-1) were significantly higher in parenterally vs. enterally fed piglets. Total small intestinal LPH activities were lower in parenterally vs. enterally fed animals. The abundance of LPH mRNA relative to elongation factor-1 alpha mRNA was not different between groups. The fractional synthesis rate of total mucosal protein and LPH was significantly lower in parenterally fed animals (67 +/- 7 and 66 +/- 7%/day, respectively) than in enterally fed animals (96 +/- 7 and 90 +/- 6%/day, respectively). The absolute synthesis rate (the amount of protein synthesized per gram of mucosa) of total mucosal protein was significantly lower in parenterally fed than in enterally fed piglets. However, the absolute synthesis rate of LPH was unaffected by the route of nutrient administration. These results suggest that the small intestine partially compensates for the effects of parenteral feeding by maintaining the absolute synthesis rate of LPH at the same levels as in enterally fed animals.

Investigation of three doses of oral insulin-like growth factor-I on jejunal lactase phlorizin hydrolase activity and gene expression and enterocyte proliferation and migration in piglets

In a previous study, oral IGF-I at 65 nM increased lactase phlorizin hydrolase (LPH) activity and villus height in piglets, however, the mechanisms were unknown. Herein, the response to a range of doses of IGF-I was investigated and we hypothesized that LPH and villus height would respond to oral IGF-I in a dose-dependent manner. Two 14-d experiments were conducted using cesarean-derived piglets. In experiment 1, piglets (n = 28) were fed formula containing 0, 33, 65, or 131 nmol/L (0, 0.25, 0.5, or 1.0 mg/L) recombinant human IGF-I. In experiment 2, 5′-bromodeoxyuridine was administered to piglets fed formula alone (n = 4) or containing 131 nmol/L IGF-I (n = 4). IGF-I did not affect body weight gain or intestinal weight or length. Jejunal villus height and LPH activity were significantly greater in piglets fed 131 nmol IGF-I/L than control piglets. Villus height and lactase activity in piglets fed the 33 and 65 nmol/L IGF-I doses were similar and intermediate between control and 131 nmol IGF-I/L. Jejunal mRNA expression and LPH polypeptide abundance were investigated in piglets receiving 0 or 131 nmol/L IGF-I. Steady state LPH mRNA abundance was significantly higher (p < 0.05) in IGF-I-treated piglets. The relative abundance of proLPHh was not significantly increased (p = 0.06) by IGF-I treatment. Mucosal DNA content and DNA synthesis were greater in piglets receiving 131 nmol IGF-I/L than control, however, enterocyte migration and mucosal protein content were unaffected. Thus, oral IGF-I increased jejunal LPH activity and LPH mRNA abundance and stimulated intestinal cell hyperplasia in normal piglets.

Effect of oral insulin on lactase activity, mRNA, and posttranscriptional processing in the newborn pig.

Insulin, found in human and pig colostrum and mature milk, appears to influence small intestinal growth and development. Ileal lactase activity is increased when porcine insulin is added to feedings administered to newborn piglets. We studied 2-day-old miniature piglets to determine whether the increase in lactase activity is accompanied by changes in enterocyte expression of lactase activity, steady-state levels of lactase mRNA, and/or posttranscriptional changes in lactase processing. We randomized the piglets to receive bottle feedings of a swine-weaning milk formula with (group F + I) or without (group F) the addition of 85 mU/ml of regular porcine insulin. The piglets were fed for 6 days (to 8 days of age), after which they were killed and the small intestine removed for analysis. Despite large differences between groups in enterocyte expression of lactase activity in the ileum, no differences were noted in the level of ileal lactase mRNA. The relative proportions of the 207, 210, and 230 kDa precursors of the 160 kDa mature lactase protein were similar between groups. These data indicate that the insulin-induced increased expression of ileal lactase activity is not regulated at the level of its mRNA or at the level of processing of the polypeptide.

Protein kinetics determined in vivo with a multiple-tracer, single-sample protocol: application to lactase synthesis

Precise analysis of the kinetics of protein/enzyme turnover in vivo has been hampered by the need to obtain multiple tissue samples at different times during the course of a continuous tracer infusion. We hypothesized that the problem could be overcome by using an overlapping (i.e., staggered) infusion of multiple stable amino acid isotopomers, which would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning over) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overlapping infusion of [2H3]leucine, [13C1]leucine, [13C1]phenylalanine, [2H5]phenylalanine, [13C6]phenylalanine, and [2H8]phenylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-to-tracee ratios of each isotopomer were measured in the plasma and jejunal free amino acid pools as well as in purified LPH polypeptides. These values were used to estimate kinetic parameters in vivo using a linear steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 +/- 1.1%/min, 17.4 +/- 11%/min, and 0.089 +/- 0.02%/min, respectively. We conclude that this multiple-tracer, single-sample protocol is a practicable approach to the in vivo measurement of protein fractional synthesis rates when only a single tissue sample can be obtained. This method has broad application and should be particularly useful for studies in humans.Precise analysis of the kinetics of protein/enzyme turnover in vivo has been hampered by the need to obtain multiple tissue samples at different times during the course of a continuous tracer infusion. We hypothesized that the problem could be overcome by using an overlapping (i.e., staggered) infusion of multiple stable amino acid isotopomers, which would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning over) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overlapping infusion of [2H3]leucine, [13C1]leucine, [13C1]phenylalanine, [2H5]phenylalanine, [13C6]phenylalanine, and [2H8]phenylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-to-tracee ratios of each isotopomer were measured in the plasma and jejunal free amino acid pools as well as in purified LPH polypeptides. These values were used to estimate kinetic parameters in vivo using a linear steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 ± 1.1%/min, 17.4 ± 11 %/min, and 0.089 ± 0.02 %/min, respectively. We conclude that this multiple-tracer, single-sample protocol is a practicable approach to the in vivo measurement of protein fractional synthesis rates when only a single tissue sample can be obtained. This method has broad application and should be particularly useful for studies in humans.

Orally administered lactoferrin increases hepatic protein synthesis in formula-fed newborn pigs

Lactoferrin is a polypeptide which is abundant in colostrum; however, its biologic effect in the neonate is unknown. The objective was to determine the potentially anabolic effect of orally administered lactoferrin on visceral organ growth and protein synthesis in newborn pigs. We studied a total of 18 unsuckled newborn pigs from six litters. Three pigs from each litter were randomly assigned to one of three dietary treatment groups (n = 6) and bottle-fed (10 mL/h) formula, formula containing physiologic levels (1 mg/mL) of added bovine lactoferrin (bLF), or colostrum. After 24 h of feeding, we measured visceral organ protein synthesis in vivo using a flooding dose of [3H]phenylalanine. We also measured visceral organ protein and DNA mass, as well as intestinal hydrolase activities and villus morphology. Hepatic protein synthesis in pigs fed either formula containing bLF or colostrum was similar and in both groups was significantly higher than in pigs fed formula. Splenic protein synthesis was not significantly different in pigs fed either formula or formula containing bLF, but was significantly higher in colostrum-fed animals. There were no significant differences in small intestinal growth, protein synthesis, or hydrolase activities between newborn pigs fed formula, formula containing bLF, or colostrum. Our results demonstrate that feeding formula containing physiologic concentrations of added bLF increased hepatic protein synthesis in newborn pigs, suggesting that colostrumborne lactoferrin serves an anabolic function in neonates.

Developmental changes in lactase-phlorizin hydrolase precursor isoforms in the rat.

Summary The purpose of this study was to determine whether the developmental decline in lactase specific activity ((μml mol/min/g protein) in the rat was associated with (a) changes in the relative quantities of immunoisolated precursor and mature forms of the enzyme purified by SDS-PAGE and/or (b) immunohistologic changes in the jejunal mucosa. We studied 10− and 16-day-old suckling rat pups, 22-day-old weaned rat pups, and adult female rats (nongravid, pregnant, and lactating). Lactase activity was three- to fourfold higher in 10-day-old pups than in adult rats. Lactase activity was 27% greater in lactating compared with nongravid or pregnant rats. Three molecular forms of the enzyme that migrated identically in all animals were observed on SDS-polyacrylamide gels stained with Coomassie blue: 140-kDa (mature brush border form), 200-kDa, and 220-kDa (apparent precursor forms). There was a striking difference in the proportions of the three polypeptides at different ages that was unrelated to animal status, i.e., pregnant or lactating. As the animals aged, the relative amount of the 140-kDa band declined from 86 ± 1.1% of the total immunoprecipitated lactase in 10-day old suckling pups to 68 ± 0.7% in adults. Simultaneously, the relative concentration of the 200-kDa band rose from 1.7 ± 0.4% in the 10-day-old to 19 ± 0.6% in adults. The relative concentration of the 220-kDa polypeptide did not change as a function of age. In vivo radiolabel infusion studies using [3H]phenylalanine demonstrated that the isotope was first incorporated into the 200-kDa band and later into the 140-kDa band. Thyroxine treatment induced a precocious decrease in lactase activity and an increase in the relative concentration of the 200-kDa precursor in the 16-day-old, but not the 10-day-old pup. Immunofluorescent microscopy demonstrated lactase in the brush border membranes of all animals. No staining of the crypt cells was observed in 10-day-old animals. By 16 days of age, faint crypt staining was seen, intensifying in the older animals.

Intestinal protein and LPH synthesis in parenterally fed piglets receiving partial enteral nutrition and enteral insulinlike growth factor 1.

Background Providing partial enteral nutrition (PEN) supplemented with insulinlike growth factor-1 (IGF-1) to parenterally fed piglets increases lactase–phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF-1 up-regulates LPH activity. Methods Newborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF-1 (1.0 mg · kg−1 · d−1) for 7 days. On day 7, [2H3]-leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis. Results Weight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross-sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P <0.05). Insulinlike growth factor-1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P < 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]-leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor-1 treatment increased total mucosal protein synthesis (60%, P < 0.05) but not LPH synthesis compared with the other two groups. Conclusions Because IGF-1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF-1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Fish oil supplementation does not impair the gut immune response to Trichinella spiralis infection in rats.

BACKGROUND Fish oil has been recommended as a source of omega-3 fatty acids for preterm infants and for therapy of some inflammatory diseases. METHODS Because fish oil supplementation could downregulate the hosts immune response, we studied the gut inflammatory response to an enteric infection in 72 rats assigned to three dietary groups with differing fatty acid profile: 1) fish oil, rich in eicosapentaenoic and docosahexaenoic acid; 2) olive oil, containing 71% monounsaturated fat; and 3) rat chow, containing 57% saturated fat. One half (n = 36) of the rats were infected with Trichinella spiralis larvae; the other half served as controls. The inflammatory response to initial infection (study 1), and type I hypersensitivity response to a subsequent parasite-derived antigenic challenge (study 2) were assessed. Jejunal inflammatory cell infiltrate, mean villus height, disaccharidase levels, changes in short-circuit current in response to glucose absorption, and chloride secretagogues (study 1) were measured 9 days after infection. Short-circuit current changes induced by chloride secretion were measured when the proximal jejunum was challenged with T. spiralis-derived antigen 40 days after infection (study 2). RESULTS In study 1, jejunal tissue from infected animals had more eosinophilic infiltrate, lower disaccharidase levels, and less glucose absorptive and chloride secretory capacity than tissue from noninfected animals. In study 2, the jejunum of infected animals showed an antigen-induced chloride secretory response, whereas no response was obtained from jejunal tissue from noninfected animals. Type of diet did not affect the response in either study. CONCLUSION Under the conditions of this experiment, fish oil supplementation did not interfere with the local intestinal inflammatory response after T. spiralis infection.

Regulation of Intestinal Apolipoprotein A-IV and C-III Expression by Dietary Lipid in Weanling Piglets

Regulation of Intestinal Apolipoprotein A-IV and C-III Expression by Dietary Lipid in Weanling Piglets

POST-TRANSLATIONAL MODIFICATION OF LACTASE-PHLORIZIN HYDROLASE DURING DEVELOPMENT

Lactase specific activity(SA) rises immediately after birth in the rat, reaches a peak at 9-12 d of age and declines to adult levels shortly after weaning. These studies were intended to determine the relationship of mature(L) to precursor(ProL) forms of lactase in suckling, weanling and adult Sprague-Dawley rats. Homogenized jejunal mucosa was used for measurement of enzyme SA (μmol/min/g protein) and for immunoisolation and purification of L and ProL. The relative quantities of each polypeptide were determined by densitometry. Samples were also treated with Endo H and Endo F. Enzyme SA and distribution are shown below.All bands were sensitive to Endo F treatment, none to Endo H. These studies demonstrate that: 1)the decrease in lactase SA in adult rats is associated with an increase in the level of a 200 kDa protein, 2) these changes occur at weaning when lactase SA is decreasing and may be partially responsible for lower SA seen in older animals. We speculate that these age-related changes may result from an alteration in a post-translational event.