Review: What have we learned about the effects of heat stress on the pig industry?

Pig production faces seasonal fluctuations. The low farrowing rate of sows mated in summer, increased carcass fatness of progeny born to the sows mated in summer, and slower growth rate of finisher pigs in summer are three economically important impacts identified in the pig industry. The purpose of this review is to examine advances over the past decade in understanding the mechanisms underlying the three impacts associated with summer conditions, particularly heat stress (HS), and to provide possible amelioration strategies. For impact 1, summer mating results in low farrowing rates mainly caused by the high frequency of early pregnancy disruptions. The contributions of semen DNA damage, poor oocyte quality, local progesterone concentrations, and suboptimal embryonic oestrogen secretion are discussed, as these all may contribute to HS-mediated effects around conception. Despite this, it is still unclear what the underlying mechanisms might be and thus, there is currently a lack of commercially viable solutions. For impact 2, there have been recent advances in the understanding of gestational HS on both the sow and foetus, with gestational HS implicated in decreased foetal muscle fibre number, a greater proportion of lighter piglets, and increased carcass fatness at slaughter. So far, no effective strategies have been developed to mitigate the impacts associated with gestational HS on foetuses. For impact 3, the slowed growth rate of pigs during summer is one reason for the reduced carcass weights in summer. Studies have shown that the reduction in growth rates may be due to more than reductions in feed intake alone, and the impaired intestinal barrier function and inflammatory response may also play a role. In addition, it is consistently reported that HS attenuates fat mobilisation which can potentially exacerbate carcass fatness when carcass weight is increased. Novel feed additives have exhibited the potential to reduce the impacts of HS on intestinal barrier function in grower pigs. Collectively, based on these three impacts, the economic loss associated with HS can be estimated. A review of these impacts is warranted to better align the future research directions with the needs of the pig industry. Ultimately, a better understanding of the underlying mechanisms and continuous investments in developing commercially viable strategies to combat HS will benefit the pig industry.

Porcine somatotropin alters insulin response in growing pigs by reducing insulin sensitivity rather than changing responsiveness.

Exogenous porcine somatotropin (pST) treatment consistently improves growth performance and reduces fat deposition in pigs, and it is hypothesized that one component of the mechanism is through altering the sensitivity and/or responsiveness to insulin. Therefore, a study was conducted to investigate the effect of pST treatment on whole-body glucose metabolism in response to varying doses of insulin. Eight barrows were surgically prepared with indwelling catheters and randomly assigned to one of two treatment groups (0 or 120 µg pST/kg BW · d) for 13 d. Whole-body glucose kinetics were measured during infusion of [6-(3)H]-glucose under basal conditions and during hyperinsulinemic-euglycemic clamps at various insulin infusion rates (7, 28, and 140, and 14, 70, and 280 ng insulin/kg BW · min) and alterations in the dose-response parameters were calculated with nonlinear regression. Treatment with pST increased basal plasma concentrations of glucose (36%; P = 0.005), insulin (276%; P = 0.001), and NEFAs (177%; P = 0.01) and decreased the rate of glucose disappearance (-59%; P = 0.001). The responsiveness (maximum response) for steady state glucose infusion rate to maintain glycemia was not altered by pST (112 vs 106 µmol/min · kg; P = 0.78), whereas the sensitivity (effective dose at 50% of maximum response) was increased almost 7-fold (1.3 vs 8.7 ng/mL; P = 0.027). Similar responses were observed for rate of glucose disappearance and insulin-dependent glucose utilization. Therefore, pST-induced insulin resistance with regard to whole-body glucose uptake is due to a reduced sensitivity to insulin, rather than a change in responsiveness.

Acute experimental mastitis is not causal toward the development of energy-related metabolic disorders in early postpartum dairy cows.

Twenty Holstein cows in early lactation (7 d in milk) were administered 100 microg of Escherichia coli lipopolysaccharide (LPS) dissolved in 10 mL of sterile 0.9% NaCl saline (treatment; TRT) or 10 mL of sterile saline (control) into both right mammary quarters to test the hypothesis that acute experimental mastitis would have negative impacts on aspects of energy metabolism that might lead to the development of metabolic disorders. A primed continuous intravenous infusion (14-micromol/kg of BW priming dose; 11.5-micromol/kg of BW per h continuous infusion) of 6,6-dideuterated glucose was used to determine pre- and posttreatment glucose kinetics using steady-state tracer methodologies. The LPS-treated cows displayed productive, clinical, and physiological signs of moderate to severe inflammation; control cows displayed no signs of immune activation. Pretreatment glucose rates of appearance (Ra) into plasma were similar (715 and 662 +/- 33 mmol/h for TRT and control, respectively) between treatment groups. Intramammary LPS infusion into TRT cows resulted in increased glucose Ra relative to control cows (mean glucose Ra from 150 through 270 min after intramammary infusion were 815 and 674 +/- 21 mmol/h for TRT and control cows, respectively). Furthermore, plasma concentrations of glucose increased, whereas plasma nonesterified fatty acids, glycerol, and beta-hydroxybutyrate concentrations decreased, in TRT relative to control cows. Interestingly, plasma insulin concentration increased dramatically in TRT cows and occurred prior to the small increase in plasma glucose concentration. Although these results only represent the early stages of inflammation, they are not consistent with a causal relationship between mastitis and energy-related metabolic disorders and instead suggest a coordinated protective effect by the immune system on metabolism during the early stages of mammary insult.

Localisation of glucose transport in the ruminant placenta: implications for sequential use of transporter isoforms.

The facilitative glucose transporters 1 and 3 are the major routes for glucose transport across placental membranes. Using light and electron microscope immunocytochemistry on acrylic sections this study shows a similar pattern of expression from mid to late pregnancy in all four ruminants examined [cow, deer, ewe and goat]. GT1 and GT3 are localised on different membrane layers of the synepitheliochorial placental barrier and glucose must utilise both isoforms sequentially to pass from the maternal to fetal circulations. It is suggested that this arrangement is designed to support the high glucose utilisation by the multilayered placenta in the ruminant.

Effects of birth weight and postnatal nutrition on neonatal sheep: IV. Organ growth.

This study investigated effects of birth weight and postnatal nutrition on organ growth in neonatal lambs. Suffolk x (Finnsheep x Dorset) low- (mean +/- SD 2.29 +/- 0.34 kg, n = 28) and high- (4.84 +/- 0.45 kg, n = 20) birth-weight male lambs were studied. Lambs within each birth weight category were allocated to be individually grown rapidly (ad libitum fed, ADG 337 g, n = 20) or slowly (ADG 150 g, n = 20) on a liquid diet to live weights up to approximately 20 kg. All organs weighed less at birth in small than in large newborns (P < 0.001), except the adrenals (P = 0.10). At birth, as a percentage of empty body weight (EBW), small newborns had larger testes (0.14 vs. 0.10%, P = 0.023) and smaller thymus (0.17 vs. 0.37%, P = 0.009), and tended to have a larger heart (0.85 vs. 0.75%, P = 0.060) and a smaller spleen (0.10 vs. 0.14%, P = 0.054) than large newborns. During the first 2 to 3 wk postpartum, small newborns had greater fractional growth rates of organs than large newborns, most notably spleen, thymus, and liver. Postnatal growth of organs was more closely associated with EBW than age, except for lungs, testes, and stomach. At completion of rearing to 20 kg of live weight, small newborns had a spleen approximately 30% heavier than large newborns (P < 0.001). Testes weights were 37% and 24% greater in small newborns reared slowly and rapidly, respectively, compared with their high-birth-weight counterparts (P = 0.034). It was also evident that postnatal nutrition altered the mass of individual organs at the conclusion of the rearing period without affecting the combined weight of dissected organs. Slowly reared lambs had a larger pancreas (+27%, P = 0.002), stomach complex (+83%, P < 0.001), large intestine (+39%, P < 0.001), entire gastrointestinal tract (+18%, P = 0.002), and testes (+54%, P = 0.016) and tended to have a larger heart (+6%, P = 0.068) than their rapidly reared counterparts at 20 kg of live weight. Rapidly reared lambs had a larger thymus (+61%, P = 0.003), liver (+34%, P < 0.001), kidneys (+33%, P < 0.001), and small intestine (+17%, P < 0.001) and tended to have a larger thyroid (+13%, P = 0.054) at 20 kg of live weight than slowly reared lambs. The functional significance of the smaller thymus at birth and increase in spleen and testes weights at 20 kg of live weight in low- compared with high-birth-weight lambs warrants further investigation. It also remains to be established whether these differences at 20 kg of live weight persist.

Determination of the systematic and random measurement error in an LC-FTICR mass spectrometry analysis of a partially characterized complex peptide mixture.

In high-throughput proteomics, a promising approach presently being explored is the use of liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) to provide measurements of the masses of tryptic peptides in complex mixtures, which can then be used to identify the proteins which gave rise to those peptides. In order to apply this method, it is necessary to account for any systematic measurement error, and it is useful to have an estimate of the random error in measured masses. In this investigation, a complex mixture of peptides derived from a partially characterized sample was analyzed by LC-FTICR-MS. Through the application of a Bayesian probability model of the data, partial knowledge of the composition of the sample is sufficient both to determine any systematic error and to estimate the random error in measured masses.

Consequences of intra-uterine growth retardation for postnatal growth, metabolism and pathophysiology.

Intra-uterine growth retardation (IUGR), caused by maternal undernutrition or placental insufficiency, is usually associated with disproportionately large reductions in the growth of some fetal organs and tissues (thymus, liver, spleen, thyroid) and impaired cellular development of other tissues (small intestine, secondary wool follicles, skeletal muscle). Growth of other tissues, most notably brain, is relatively unimpaired. In our restudy of postnatal consequences of IUGR in the offspring of prolific ewes, growth-retarded newborn lambs tended to be hypoglycaemic and showed sluggish postnatal engagement of the growth hormone (GH)-insulin-like growth factor (IGF) system. When artificially reared in an optimum environment, low birth weight lambs grew at rates similar to those of normal lambs. However, low birth weight lambs were fatter at any given weight, apparently related to their high energy intakes, especially soon after birth, had low maintenance energy requirements, and limited capacity for bone and muscle growth. These growth characteristics were accompanied by higher plasma concentrations of GH and leptin, and lower concentrations of insulin-like growth factor I (IGF-I) during the first 2 weeks of postnatal life, and higher concentrations of insulin during subsequent growth up to 20 kg body weight. Emerging evidence indicates that in sheep, as in rodents, fetal programming of postnatal cardiovascular and metabolic dysfunctions is associated with IUGR and may be mediated partly by overexposure of the fetus to cortisol. Similar postnatal responses can be elicited by maternal undernutrition or cortisol treatment in early to mid-pregnancy without changing the growth of the fetus or placenta.

Effects of birth weight and postnatal nutrition on neonatal sheep: III. Regulation of energy metabolism.

This study investigated effects of birth weight and postnatal nutrition on regulation of energy metabolism in the neonatal lamb. Low (mean +/- SD 2.289 +/- 0.341 kg, n = 28) and high (4.840 +/- 0.446 kg, n = 20) birth weight male Suffolk x (Finnsheep x Dorset) lambs were individually reared on a liquid diet to grow rapidly (ad libitum fed, ADG = 337 g, n = 20) or slowly (ADG = 150 g, n = 20) from birth to live weights (LW) up to approximately 20 kg. At birth, small newborns had higher plasma concentrations of urea nitrogen (mean +/- SEM 8.31 +/- 0.25 vs 6.39 +/- 0.32 mM, P = 0.002) and somatotropin (ST, 49.1 +/- 17.0 vs 10.8 +/- 4.3 ng/mL, P = .045) and lower IGF-I (36.1 +/- 6.8 vs 157.7 +/- 21.8 ng/mL, P < 0.001) than large newborns. Plasma glucose (1.42 +/- 0.23 vs 2.63 +/- 0.95 mM, P = 0.147) and insulin (0.09 +/- 0.02 vs 0.13 +/- 0.06 ng/mL, P = 0.264) concentrations did not differ. Urea nitrogen concentration in plasma peaked and then declined rapidly in all lambs during the first week postpartum, and plasma ST declined on a body-weight-related basis from birth. During rearing to 20 kg LW, plasma insulin was higher in low- vs high-birth-weight lambs. Lambs fed ad libitum had greater plasma concentrations of glucose, urea nitrogen, insulin, and IGF-I compared to those fed a restricted diet (ADG = 150 g). The results suggest that during the early postpartum period, newborn lambs exhibit the fetal characteristic of high rates of amino acid oxidation. The results also support the notion that, at birth, low-birth-weight lambs are less mature than high-birth-weight lambs in aspects of metabolic and endocrine development, which may enhance their capacity to utilize amino acids for energy production and to support gluconeogenesis during the immediate postpartum period. Being small at birth also resulted in elevated plasma insulin concentrations when adequate nutriment to support moderate or rapid growth was provided postpartum, although it remains to be elucidated whether this more chronic effect persists in the longer term.

Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.

Dairy cows suffer from an intense energy deficit at parturition due to the onset of copious milk synthesis and depressed appetite. Despite this deficit, maternal metabolism is almost completely devoted to the support of mammary metabolism. Evidence from rodents suggests that, during periods of nutritional insufficiency, a reduction in plasma leptin serves to co-ordinate energy metabolism. As an initial step to determine if leptin plays this role in periparturient dairy cows, changes in the plasma concentration of leptin were measured during the period from 35 days before to 56 days after parturition. The plasma concentration of leptin was reduced by approximately 50% after parturition and remained depressed during lactation despite a gradual improvement in energy balance; corresponding changes occurred in the abundance of leptin mRNA in white adipose tissue. To determine whether negative energy balance caused this reduction in circulating leptin, cows were either milked or not milked after parturition. Absence of milk removal eliminated the energy deficit of early lactation, and doubled the plasma concentration of leptin. The plasma concentration of leptin was positively correlated with plasma concentrations of insulin and glucose, and negatively correlated with plasma concentrations of growth hormone and non-esterified fatty acids. In conclusion, the energy deficit of periparturient cows causes a sustained reduction in plasma leptin. This reduction could benefit early lactating dairy cows by promoting a faster increase in feed intake and by diverting energy from non-vital functions such as reproduction.

Maternal leptin is elevated during pregnancy in sheep.

Maternal plasma leptin is elevated during pregnancy in several species, but it is unclear to what extent this elevation reflects changes in adiposity or energy balance. Therefore, Karakul ewes (n = 8) were fed to minimize changes in maternal energy status over the pregnancy-lactation cycle. They were studied 20-40 d before breeding and during mid pregnancy (d 50-60 post coitus [PC]), late pregnancy (d 125-135 PC) and early lactation (d 15-22 post partum). Consistent with the maintenance of near energy equilibrium in nongravid maternal tissues, maternal body weight was increased only during late pregnancy when the weight of the conceptus became significant and plasma concentrations of insulin, NEFA and glucose did not vary with physiological state. In contrast, maternal plasma leptin concentration rose from 5.3 to 9.5 ng/mL between prebreeding and mid pregnancy and then declined progressively through late pregnancy and early lactation. Leptin gene expression increased 2.3 fold in maternal white adipose tissue (WAT) from prebreeding to mid pregnancy and declined to prebreeding levels during early lactation. To determine whether tissue response to insulin was involved in this effect, insulin tolerance tests were performed. The maternal plasma glucose response declined from prebreeding to early lactation, but was not correlated with either plasma leptin concentration or WAT leptin mRNA abundance. In conclusion, pregnancy causes an increase in the synthesis of leptin in sheep. This stimulation does not require increases in adiposity or energy balance and is unrelated to the ability of insulin to promote glucose utilization.

Proteomics characterization of abundant Golgi membrane proteins.

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Nutritional regulation of the genes encoding the acid-labile subunit and other components of the circulating insulin-like growth factor system in the sheep.

In sheep, perinatal maturation of the endocrine arm of the insulin-like growth factor (IGF) system is characterized by two developmental events. First, concentrations of circulating IGF-I increase rapidly after birth and become responsive to changes in nutrition and growth hormone (GH). Second, the liver initiates synthesis of a serum protein called the acidlabile subunit (ALS). The acid-labile subunit promotes the endocrine actions of IGF-I and -II by recruiting them to long-lived complexes of 150 kDa. In this study, we examined the effect of nutrition on hepatic expression of the ALS gene around the time of birth and later in life. Expression of genes encoding other components of the circulating IGF system was also measured. At d 130 of fetal life, fetuses suffering from chronic undernutrition caused by placental insufficiency had lower expression of the ALS and IGF-I genes than well-nourished fetuses, but they did not have any changes in the expression of the IGF-binding protein (IGFBP)-2 or IGFBP-3 genes. In early postnatal life, hepatic gene expression was analyzed between d 12 and 38 in lambs fed a milk replacer at levels sustaining weight gains of 150 or 337 g/d. The lower plane of nutrition decreased the expression of the ALS, IGF-I, and GH receptor genes and increased the expression of the IGFBP-2 gene; expression of the IGFBP-3 gene was not affected by nutrition at this stage of life. Finally, hepatic gene expression was measured in 3-mo-old lambs offered ad libitum levels of a balanced diet or of a diet limiting for both energy and protein. Although the rate of growth of the lambs fed the limiting diet was reduced by 38%, the only effect detected in hepatic gene expression was a ninefold increase in the abundance of IGFBP-2 mRNA. Overall, these results indicate that undernutrition during late fetal and early postnatal life delays hepatic expression of the ALS gene and final maturation of the endocrine IGF system.

Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep.

Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of (125)I-labeled bovine leptin was linearly related (R(2)= 0.99) to the log of added bovine or ovine leptin between 0.1 to 2.0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of! nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.

Protein nutrition in late pregnancy, maternal protein reserves and lactation performance in dairy cows.

Empirical evidence suggests that prolonged underfeeding of protein to late-pregnant dry cows can have modest negative carry-over effects on milk volume and/or protein yield during early lactation, and may also cause increased incidence of metabolic diseases associated with fatty liver. However, assessment of requirements is hampered by lack of information on relationships between dietary intake of crude protein (N x 6.25) and metabolizable protein supply during late pregnancy, and by incomplete understanding of the quantitative metabolism of amino acids in maternal and conceptus tissues. Inability of the postparturient cow to consume sufficient protein to meet mammary and extra-mammary amino acid requirements, including a significant demand for hepatic gluconeogenesis, necessitates a substantial, albeit transient, mobilization of tissue protein during the first 2 weeks of lactation. Ultimately, much of this mobilized protein appears to be derived from peripheral tissues, especially skeletal muscle and, to a lesser extent, skin, through suppression of tissue protein synthesis, and possibly increased proteolysis. In the shorter term, soon after calving, it is likely that amino acids required for hepatic glucose synthesis are diverted from high rates of synthesis of splanchnic tissue and export proteins, including serum albumin. The prevailing endocrine milieu of the periparturient cow, including major reductions in plasma levels of insulin and insulin-like growth factor-I, together with insulin resistance in peripheral tissues, must permissively facilitate, if not actively promote, net mobilization of amino acids from these tissues.

Mass spectrometry of native rat amelogenins: primary transcripts, secretory isoforms, and C-terminal degradation.

Cloning technologies have established unambiguously that amelogenins always seem larger in molecular weight (Mr) by gel electrophoresis (SDS-PAGE) than by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in vivo. In this study, discrete protein fractions at 31-20 kDa (Mr(SDS)) were prepared from freeze-dried rat incisor enamel by techniques optimized for preserving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amelogenin. As expected, the molecular weights estimated by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-PAGE. Plots of Mr(SDS) vs. Mr(MS) for all fractions showed high linear correlation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform of rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major proteins forming the 25-, 24-, and 23-kDa fractions (Mr(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P169-A170, P164-L165, and F151-S152 C-terminal cleavage sites, respectively. Proteins in the 20-kDa fraction (Mr(SDS)) most closely matched by ESI-MS fragments of the R156 secretory isoform that were C-terminally-modified at the equivalent P164-L165 site.

Organization and regulation of the gene encoding the sheep acid-labile subunit of the 150-kilodalton insulin-like growth factor-binding protein complex.

In adult animals, most circulating insulin-like growth factor I (IGF-I) and IGF-II is sequestered in a 150-kDa complex composed of 1 molecule each of IGF, IGF-binding protein-3 or -5, and the acid-labile subunit (ALS). Capture of IGF in ALS-containing complexes increases their circulating half-lives and concentrations and suppresses their hypoglycemic potential. ALS has been studied almost exclusively in rodents and primates, and no information exists in the sheep despite its extensive use to study the circulating IGF system. To initiate studies in the sheep, we isolated the ovine ALS gene and characterized its spatial and developmental regulation. The ALS gene spans about 3.0 kb of chromosomal DNA and consists of 2 exons interrupted by a 977-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide; exon 2 encodes the remaining 27 amino acids of the signal peptide and the entire mature protein of 579 amino acids. Transcription initiation occurs at nucleotides -58 and -29 (ATG, + 1), 2 sites that are not preceded by TATA or initiator sequences. A DNA fragment extending from -727 to - 11 of the sheep ALS gene directed basal expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. GH increased promoter activity by 1.8-fold, consistent with conservation in the sheep promoter of the GH response element previously identified in the mouse gene. A survey of adult tissues by Northern analysis revealed the presence of a 2.2-kb transcript only in liver. Weak expression was first detected in liver on day 130 of fetal life, increased suddenly on day 7 of postnatal age, and changed little thereafter. The sheep is a useful model to understand the regulation and role of ALS, particularly around the time of birth, when final maturation of the circulating IGF system occurs.

Effects of birth weight and postnatal nutrition on neonatal sheep: II. Skeletal muscle growth and development.

This study investigated effects of birth weight and postnatal nutrition on growth and development of skeletal muscles in neonatal lambs. Low (L; mean +/- SD 2.289 +/- .341 kg, n = 28) and high (H; 4.840 +/- .446 kg, n = 20) birth weight male Suffolk x (Finnsheep x Dorset) lambs were individually reared on a liquid diet to grow rapidly (ad libitum fed, ADG 337 g, n = 20) or slowly (ADG 150 g, n = 20) from birth to live weights (LW) up to approximately 20 kg. At birth, weight of semitendinosus (ST) muscle in L lambs was 43% that in H lambs; aggregate weights of ST and seven other dissected muscles were similarly reduced. In ST muscle of L lambs, mass of DNA, RNA, and protein were also significantly reduced to levels 67, 60, and 34%, respectively, of those in H lambs. However, myofiber numbers of ST, tibialis caudalis, or soleus muscles did not differ between the L and H birth weight lambs and did not change during postnatal growth. During postnatal rearing, daily accretion rate of dissected muscle was lower in L than in H lambs. Accretion of muscle per kilogram of gain in empty body weight (EBW) was reduced in the slowly grown L lambs compared with their H counterparts, although the difference was less pronounced between the rapidly grown L and H lambs. Throughout the postnatal growth period, ST muscle of L lambs contained less DNA with a higher protein:DNA ratio at any given muscle weight than that of H lambs. Slowly grown lambs had heavier muscles at any given EBW than rapidly grown lambs. Content of DNA and protein:DNA ratio in ST muscle were unaffected by postnatal nutrition, but RNA content and RNA:DNA were greater and protein:RNA was lower at any given muscle weight in rapidly grown lambs. Results suggest that myofiber number in fetal sheep muscles is established before the presumed, negative effects of inadequate fetal nutrient supply on skeletal muscle growth and development become apparent. However, proliferation of myonuclei may be influenced by fetal nutrition in late pregnancy. Reduced myonuclei number in severely growth-retarded newborn lambs may limit the capacity for postnatal growth of skeletal muscles.

Influences on fetal and placental weights during mid to late gestation in prolific ewes well nourished throughout pregnancy.

This study investigated associations between fetal and placental weights from 85 to 130 days gestation in 49 fetuses from 21 ewes of a prolific genotype used as an experimental model of intrauterine growth retardation. The proportion of variation in fetal weight explained by placental weight increased from zero at 85 days to 91% (residual standard deviation (RSD) = 260 g) at 130 days. Overall, stage of pregnancy plus placental weight accounted for 96% of fetal weight variation (RSD = 212 g). Litter size and number of fetuses per uterine horn also influenced individual fetal weights. Gestational age, litter size, placental weight per ewe, and liveweight and condition score of ewes during early to mid gestation (initial LW and CS) explained 99.5% of the variation in fetal weight per ewe (RSD = 236 g). Most variation (86%) in placental weight was explained by stage of pregnancy, litter size, number of placentomes, and initial LW and CS (RSD = 53 g). Placental weight per ewe was influenced by stage of pregnancy, litter size and initial ewe LW and CS (R2 = 0.97; RSD = 89 g). The association of fetal and placental weights with initial ewe LW was positive, and with initial CS was negative. The results show that in the absence of overt nutritional restriction of pregnant ewes, fetal and placental weights are tightly coupled during late gestation and ewe fatness during early pregnancy is inversely related to placental and fetal weights. They demonstrate that placental weight explains most of the variation in fetal weight in the present intrauterine growth retardation model.

Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

We have investigated the relevant protease activity in rat liver, which is responsible for most of the receptor-mediated epidermal growth factor (EGF) degradation in vivo. EGF was sequentially cleaved by endosomal proteases at a limited number of sites, which were identified by high performance liquid chromatography and mass spectrometry. EGF proteolysis is initiated by hydrolysis at the C-terminal Glu(51)-Leu(52) bond. Three additional minor cleavage sites were identified at positions Arg(48)-Trp(49), Trp(49)-Trp(50), and Trp(50)-Glu(51) after prolonged incubation. Using nondenaturating immunoprecipitation and cross-linking procedures, the major proteolytic activity was identified as that of the cysteine protease cathepsin-B. The effect of injected EGF on subsequent endosomal EGF receptor (EGFR) proteolysis was further evaluated by immunoblotting. Using endosomal fractions prepared from EGF-injected rats and incubated in vitro, the EGFR was lost with a time course superimposable with the loss of phosphotyrosine content. The cathepsin-B proinhibitor CA074-Me inhibited both in vivo and in vitro the endosomal degradation of the EGFR and increased the tyrosine phosphorylation states of the EGFR protein and the molecule SHC within endosomes. The data, therefore, describe a unique pathway for the endosomal processing of internalized EGF receptor complexes, which involves the sequential function of cathepsin-B through selective degradation of both the ligand and receptor.

N-terminal sequence analysis of SAA-derivatives purified from murine inflammatory macrophages.

The pathogenesis of secondary amyloidosis in vivo is not well-understood. Experimental studies suggest that incomplete degradation of acute phase serum amyloid A (SAA), presumably endocytosed by activated monocytoid cells, may lead to intralysosomal formation of amyloid A (AA). To establish a possible link between these two events, we have carried out partial N-terminal sequence analysis of affinity purified SAA derivatives from peritoneal macrophages isolated at 4 weeks post-infection from alveolar hydatid cyst infected C57BL/6 mice. The macrophage lysates yielded five N-terminally intact SAA derivatives of approximately 5 to approximately 12 kDa which reacted with anti-mouse AA IgG, and contained a mixture of SAA1 and SAA2 isoforms. The SAA2:SAA1 ratio, evaluated from their proportion present in each M(r) SAA derivative, showed a decrease with the decreasing apparent mass of the N-terminally infected SAA material. These results not only confirm that both SAA1 and SAA2 are processed by activated monocytoid cells but, more importantly, establish a plausible link between N-terminally intact SAA derivatives and formation of AA within activated monocytoid cells.