Catarina Rippe

Age, sex, and weight at weaning influence organ weight and gastrointestinal development of weanling pigs

The present study was designed to determine the interrelationships between sex, weaning age, and weaning weight on aspects of physiological and gastrointestinal development in pigs. Forty-eight Large White x Landrace pigs were used in a factorial arrangement with the respective factors being: age at weaning ( 14 or 28 days), weight at weaning ( heavy or light), sex ( boar or gilt), and time after weaning ( 1, 7, and 14 days). At weaning, 48 pigs were removed from the sow; 16 pigs were then fasted for 24 h before euthanasia for determination of organ weights, gut histology, and enzymology, and 32 pigs were offered a high quality pelleted weaner diet ad libitum for subsequent assessment of organ weights, histology, and enzymology at 7 and 14 days after weaning. On Day 6 and 13 after weaning, 2 pigs from each group had their feed removed, and 24 h later were euthanased and similar measurements were taken. In general, the data highlighted the overall gastrointestinal underdevelopment of pigs weaned at 2 weeks of age and of pigs weaned light-for-age at either 2 or 4 weeks. Heavier body organs, gastrointestinal organs, and accessory digestive organs observed after weaning, except for the spleen, presumably reflected the increase in substrates available for cellular growth as feed intake increased after weaning, and the development of organs required to process this feed. Interestingly, the relative weights (% of liveweight) of the stomach and small intestine and, to a lesser extent, the caecum and colon, were greater in the light, 14-day-old weaned pigs, but these differences diminished with increasing time after weaning. Consistent effects due to age, weight, and sex were not observed for villous height and crypt depth, or for the specific activities of the brush-border and pancreatic enzymes measured. However, increases (P < 0.001) in the activities of maltase (P < 0.001), glucoamylase ( P < 0.001), and sucrase (P = 0.020) ( all expressed per gram of mucosa), and that of trypsin ( per gram of pancreas), occurred by 14 days after weaning. This most likely reflected the inducible nature of these enzymes in response to the increasing intake of substrates provided in the diet. In contrast, the specific activity of lactase declined (P = 0.012) in the first 14 days after weaning. These data suggest that pigs weaned at 2 weeks of age and pigs weaned light-for-age at either 2 or 4 weeks have a less developed gastrointestinal tract, and that its development after weaning might proceed differently to that of pigs weaned older and heavier. (Less)

Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat. Effects of molecular size and conformation.

Aim:  Polysaccharides and many other non‐protein polymers generally have a more open, flexible and asymmetrical structure compared with globular proteins. For a given molecular weight (MW), the Stokes–Einstein radius (ae) of the following polymers increases in the order: Ficoll < dextran ≤ pullulan < polyethylene oxide (PEO). We have tested the hypothesis that such an increase in ‘molecular extension’ will increase the molecule’s glomerular permeability. Thus, we investigated the glomerular sieving coefficients (θ) of the mentioned polymers and of the negatively charged and extended protein bikunin.

Mitochondrial ATP synthase--a possible target protein in the regulation of energy metabolism in vitro and in vivo.

Abstract The increasing prevalence of obesity in the Western world has stimulated an intense search for mechanisms regulating food intake and energy balance. A number of appetite-regulating peptides have been identified, their receptors cloned and the intracellular events characterized. One possible energy-dissipating mechanism is the mitochondrial uncoupling of ATP-synthesis from respiratory chain oxidation through uncoupling proteins, whereby energy derived from food could be dissipated as heat, instead of stored as ATP. The exact role of the uncoupling proteins in energy balance is, however, uncertain. We show here that mitochondrial F1F0-ATP synthase itself is a target protein for an anorectic peptide, enterostatin, demonstrated both after affinity purification of rat brain membranes and through a direct physical interaction between enterostatin and purified F1-ATP synthase. In insulinoma cells (INS-1) enterostatin was found to target F1F0-ATP synthase, causing an inhibition of ATP production, an increased thermogenesis and increased oxygen consumption. The experiments suggest a role of mitochondrial F1F0-ATP synthase in the suppressed insulin secretion induced by enterostatin. It could be speculated that this targeting mechanism is involved in the decreased energy efficiency following enterostatin treatment in rat.

Deletion of Dicer in Smooth Muscle Affects Voiding Pattern and Reduces Detrusor Contractility and Neuroeffector Transmission

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca2+ channels in the detrusor.

Biomechanical properties and innervation of the female caveolin‐1‐deficient detrusor

BACKGROUND AND PURPOSE Caveolin‐1‐deficiency is associated with substantial urogenital alterations. Here, a mechanical, histological and biochemical characterization of female detrusors from wild‐type and caveolin‐1‐deficient (KO) mice was made to increase the understanding of detrusor changes caused by lack of caveolae.

Arterial dysfunction but maintained systemic blood pressure in cavin-1-deficient mice.

Caveolae are omega-shaped plasma membrane micro-domains that are abundant in cells of the vascular system. Formation of caveolae depends on caveolin-1 and cavin-1 and lack of either protein leads to loss of caveolae. Mice with caveolin-1 deficiency have dysfunctional blood vessels, but whether absence of cavin-1 similarly leads to vascular dysfunction is not known. Here we addressed this hypothesis using small mesenteric arteries from cavin-1-deficient mice. Cavin-1-reporter staining was intense in mesenteric arteries, brain arterioles and elsewhere in the vascular system, with positive staining of both endothelial and smooth muscle cells. Arterial expression of cavin-1, -2 and -3 was reduced in knockout (KO) arteries as was expression of caveolin-1, -2 and -3. Caveolae were absent in the endothelial and smooth muscle layers of small mesenteric arteries as determined by electron microscopy. Arginase, a negative regulator of nitric oxide production, was elevated in cavin-1 deficient arteries as was contraction in response to the α1-adrenergic agonist cirazoline. Detailed assessment of vascular dimensions revealed increased media thickness and reduced distensibility, arguing that enhanced contraction was due to increased muscle mass. Contrasting with increased α1-adrenergic contraction, myogenic tone was essentially absent and this appeared to be due in part to increased nitric oxide production. Vasomotion was less frequent in the knock-out vessels. In keeping with the opposing influences on arterial resistance of increased agonist-induced contractility and reduced myogenic tone, arterial blood pressure was unchanged in vivo. We conclude that deficiency of cavin-1 affects the function of small arteries, but that opposing influences on arterial resistance balance each other such that systemic blood pressure in unstressed mice is well maintained.

The enzyme levels in blood are not affected by oral administration of a pancreatic enzyme preparation (Creon 10,000) in pancreas-insufficient pigs.

After oral intake, small amounts of intact protein may be absorbed into the blood circulation. The current study investigated whether orally administered pancreatic enzymes were absorbed from the intestine. The study included 28 pigs; 3 control pigs with intact pancreatic function and 25 pigs that were made exocrine pancreas insufficient by duct ligation (20 pigs) or total pancreatectomy (5 pigs).The pigs received a pancreatic enzyme preparation (0, 2, 4, or 8 g of Creon 10,000) together with the feed. The blood plasma was analyzed for pancreatic lipase activity with a [3H]-triolein substrate assay, while (pro)colipase and cationic trypsin(ogen) levels were measured with enzyme-linked immunosorbent assay (ELISA). Administration of Creon (0–8 g) caused no significant changes in plasma (pro)colipase or cationic trypsin(ogen) levels. Lipase activity peaks in plasma samples were found, but they did not correspond to the administration of Creon. The potential source of these plasma lipase activity peaks is discussed. The results showed no absorption into blood of pancreatic enzymes after oral administration (0, 2, 4, or 8 g of Creon mixed with 100 g of feed) to pancreas-insufficient pigs.

Effect of Long-term High-Fat Feeding on the Expression of Pancreatic Lipases and Adipose Tissue Uncoupling Proteins in Mice

Introduction A diet containing a high amount of fat has been shown, in short-term studies, to increase the expression of pancreatic lipase and colipase. Aim To investigate the effects of long-term high-fat–feeding (113 days) on the mRNA expression of pancreatic lipase, colipase, pancreatic lipase-related proteins (1 and 2), and uncoupling proteins during the development of obesity and glucose intolerance. Methodology Mice were fed either a high-fat or standard diet and killed after 3, 13, 57, and 113 days. Brown and white adipose tissues from the pancreas were collected for mRNA extraction. Results The high-fat–fed mice became obese and glucose-intolerant by 113 days. The high-fat diet increased lipase (p < 0.05) expression initially. At the end of the experiment, the lipase levels had decreased to the level of the control. Colipase levels did not change during the first 57 days of high-fat feeding but decreased below control levels by 113 days (p < 0.05). The high-fat diet increased brown adipose tissue uncoupling protein 1 (UCP1)(p < 0.005) expression but not the expression of uncoupling protein 2. Conclusion Long-term high-fat feeding, leading to glucose intolerance, occurs with a simultaneous decrease in the mRNA expression of pancreatic lipase and colipase and an increase in UCP1 expression.

Glomerular permeability to macromolecules in the Necturus kidney.

Many aspects of the glomerular filtration of macromolecules remain controversial, including the location of the major filtration barrier, the effects of electrical charge, and the reason the filtration barrier does not clog. We examined these issues in anesthetized Necturus maculosus, using fluorescently labeled probes and a two-photon microscope. With the high resolution of this system and the extraordinary width ( approximately 3.5 mum) of the glomerular basement membrane (GBM) in this salamander, we were able to visualize fluorescent molecules in the GBM in vivo. GBM/plasma concentration ratios for myoglobin, ovalbumin, and serum albumin did not differ from that of inulin, indicating that the GBM does not discriminate among these molecules. The GBM/plasma concentration ratios for fluoresceinated dextran 500 and 2,000 kDa were significantly below that of inulin. Glomerular sieving coefficients (GSCs) for various macromolecules decreased as molecular mass increased, and the GSCs for bovine or human serum albumin were extremely low. The effect of electrical charge on filterability of a macromolecule was also examined. The GSCs for native (anionic) and neutral human serum albumin were not significantly different, nor did GSCs for anionic and neutral dextran 40 kDa differ, indicating that charge has no detectable effect on filterability of these macromolecules. These studies indicate that the main filtration barrier to albumin is the podocyte slit diaphragm. Electron microscopic studies revealed many cell processes within the GBM. Macromolecules that penetrated the GBM were taken up by mesangial cells and endothelial cells, suggesting that these cells help to prevent clogging of the filter.

Impaired contractility and detrusor hypertrophy in cavin-1-deficient mice.

Caveolae are membrane invaginations present in a variety of cell types. Formation of caveolae depends on caveolins and on the more recently discovered family of proteins known as the cavins. Genetic ablation of caveolin-1 was previously shown to give rise to a number of urogenital alterations, but the effects of cavin-1 deletion on urogenital function remain unknown. Here we characterised detrusor contractility and structure in cavin-1-deficient mice. Electron microscopy demonstrated essentially complete lack of caveolae in the knock-out detrusor, and immunoblotting disclosed reduced levels of cavin-3 and of all caveolin proteins. Bladder weight was increased in male knock-out mice, and length-tension relationships demonstrated a reduction in depolarisation-induced contraction. Contractility in response to muscarinic receptor activation was similarly reduced. Despite these functional changes, micturition patterns were similar in conscious and freely moving animals and diuresis was unchanged. Our breeding additionally disclosed that the number of knock-out mice generated in heterozygous crosses was lower than expected, suggesting embryonic/perinatal lethality. In conclusion, this is the first study to show that cavin-1 is critical for detrusor caveolae and for the overall contractility and structure of the urinary bladder.