Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.     

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.     

Lipoprotein metabolism in the ovariectomized rat

The hyperlipoproteinemia observed after ovariectomy in rats was previously shown to be associated with increased concentrations of cholesterol, triglycerides, and apolipoproteins B, E, and C. In the present study, it was shown that increases in low density lipoproteins and high density lipoproteins were almost entirely responsible for the changes in plasma lipids and apolipoproteins after ovariectomy. The size of the low density lipoproteins and high density lipoproteins isolated from the plasma of ovariectomized rats as determined by agarose chromatography appeared to be somewhat different from that of control rats. Specifically, the apolipoprotein B appeared to be associated with somewhat smaller particles, whereas the apolipoprotein E from those rats appeared to be associated with larger particles than that of control rats. To determine the mechanism for the increased plasma low density lipoproteins, apolipoprotein B pool sizes and turnover rates were calculated and compared. In addition to an increased mass of low density lipoproteins in ovariectomized rats, the turnover rate of low density lipoproteins was increased almost twofold, indicating an increased low density lipoprotein synthesis and catabolism in those animals. We postulate that the increased low density lipoprotein levels of ovariectomized rats are due to an initial increased production of low density lipoproteins, followed by an enhanced catabolism of low density lipoproteins to establish a steady state at higher plasma low density lipoprotein concentrations.     

A moderate inflammation caused by the deterioration of housing conditions modifies Trp metabolism but not Trp requirement for growth of post-weaned piglets

Deterioration of the environment in which piglets are housed after weaning induces a moderate inflammatory response and modifies tryptophan (Trp) metabolism that can, in turn, decrease Trp availability for growth. We hypothesised that a Trp supply above the current recommendations may be required to preserve Trp availability and to maximise the growth of pigs suffering from moderate inflammation. The aim of this experiment was to compare growth performance and plasma concentrations of Trp and some of its metabolites in piglets, suffering or not from moderate inflammation, when they were fed diets containing graded levels of standardised ileal digestible (SID) Trp, obtained with the addition of crystalline l-Trp to the same basal diet (15%, 18%, 21% or 24%, relative to SID lysine). Differences in inflammatory status were obtained by housing the pigs under different sanitary conditions. Forty blocks of four littermate piglets each were selected and weaned at 4 weeks of age. The experimental design consisted of a split plot where the housing conditions (moderate inflammation v. control) were used as the main plot and dietary Trp content as the subplot. Body weight gain and feed intake were recorded 3, 5 and 7 weeks after weaning. Blood was sampled 13, 36 and 43 days after weaning to measure plasma concentrations of Trp, kynurenine and nicotinamide (i.e. two metabolites of Trp catabolism) and haptoglobin, a major acute phase protein in pigs. There was no interaction between dietary Trp and inflammatory status, irrespective of the response criterion. Compared with control pigs, pigs housed in poor housing conditions consumed less feed (P < 0.0001), had a lower growth rate (P < 0.001), higher plasma concentrations of haptoglobin (P < 0.05) and lower concentrations of plasma Trp irrespective of the Trp content in the diet. Increasing the Trp content in the diet improved feed intake (P < 0.05), growth rate and feed/gain (P < 0.05), but did not prevent the deterioration of performance induced by moderate inflammation because of poor housing conditions. The results of this study suggest that an inflammatory response caused by poor housing sanitary conditions altered Trp metabolism and growth performance, but this was not prevented by additional dietary crystalline l-Trp.     

Density gradient characterization of the high density lipoproteins in cholesterol-fed hyper- and hyporesponding patas monkeys (Erythrocebus patas)

Diet-induced changes in high density lipoprotein (HDL) density and size were studied in patas monkeys. When the animals were switched from a moderate fat-low cholesterol diet to a high fat-high cholesterol (HFHC) diet, the plasma apoA-I levels increased initially in all of the animals. The apoA-I levels remained elevated in monkeys able to maintain their plasma cholesterol concentrations near basal levels (hyporesponders), but began to decrease in monkeys who became severely hypercholesterolemic (hyperresponders), reaching levels as low as 65-70% of their basal value by 24 weeks. The larger, lipid-rich HDL (HDL2) was shown by density gradient ultracentrifugation and gradient-PAGE (polyacrylamide gel electrophoresis) to be the HDL fraction responsible for these changes in apoA-I, completely accounting for the increase in apoA-I in hyporesponders and the decrease in apoA-I in hyperresponders. The HDL3 levels remained unchanged in hyporesponders but increased markedly in hyperresponders, partially compensating for the decrease of HDL2 in those animals. Gradient-PAGE showed the HDL3 to be heterogeneous, containing at least two populations of particles of the same density but differing significantly in size. The smaller of these HDL3 were most prominent in the HFHC-fed hyperresponders. These data show that nonhuman primate HDL is both physically and metabolically heterogeneous, and indicate that a high fat-high cholesterol diet-induced hypercholesterolemia severely depresses the HDL2 levels.     

Complex Actions of Ionomycin in Cultured Cerebellar Astrocytes Affecting Both Calcium-Induced Calcium Release and Store-Operated Calcium Entry

The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca²⁺ in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca²⁺ into the cytosol, a number of studies have demonstrated that the mechanism of action is likely to be more complex, involving modulation of intrinsic Ca²⁺ signaling pathways. In the present study we assessed the effect of ionomycin on primary cultures of murine cerebellar astrocytes. Ionomycin concentrations ranging from 0.1 to 10 μM triggered a biphasic increase in cytosolic Ca²⁺, consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca²⁺, the peak phase persisted. The peak amplitude could be lowered significantly by application of dantrolene, demonstrating involvement of Ca²⁺-induced Ca²⁺-release (CICR). The plateau phase was markedly reduced when store-operated Ca²⁺-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca²⁺ stores in astrocytes, causing release of Ca²⁺ into the cytosol, which in turn triggers further depletion of the stores through CICR and subsequently activates SOCE. This mechanistic action of ionomycin is important to keep in mind when employing it as a pharmacological tool.     

Serum metabolites associated with feed efficiency in black angus steers

Introduction

Improving feed utilization in cattle is required to reduce input costs, increase production, and ultimately improve sustainability of the beef cattle industry. Characterizing metabolic differences between efficient and non-efficient animals will allow stakeholders to identify more efficient cattle during backgrounding.

Objectives

This study used an untargeted metabolomics approach to determine differences in serum metabolites between animals of low and high residual feed intake.

Methods

Residual feed intake was determined for 50 purebred Angus steers and 29 steers were selected for the study steers based on low versus high feed efficiency. Blood samples were collected from steers and analyzed using untargeted metabolomics via mass spectrometry. Metabolite data was analyzed using Metaboanalyst, visualized using orthogonal partial least squares discriminant analysis, and p-values derived from permutation testing. Non-esterified fatty acids, urea nitrogen, and glucose were measured using commercially available calorimetric assay kits. Differences in metabolites measured were grouped by residual feed intake was measured using one-way analysis of variance in SAS 9.4.

Results

Four metabolites were found to be associated with differences in feed efficiency. No differences were found in other serum metabolites, including serum urea nitrogen, non-esterified fatty acids, and glucose.

Conclusions

Four metabolites that differed between low and high residual feed intake have important functions related to nutrient utilization, among other functions, in cattle. This information will allow identification of more efficient steers during backgrounding.