Quinine is a natural molecule commonly used as a flavouring agent in tonic water. Diet supplementation with quinine leads to decreased body weight and food intake in rats. Quinine is an in vitro inhibitor of Trpm5, a cation channel expressed in taste bud cells, the gastrointestinal tract and pancreas. The objective of this work is to determine the effect of diet supplementation with quinine on body weight and body composition in male mice, to investigate its mechanism of action, and whether the effect is mediated through Trpm5.
Compared with mice consuming AIN, a regular balanced diet, mice consuming AIN diet supplemented with 0.1% quinine gained less weight (2.89 ± 0.30 g vs 5.39 ± 0.50 g) and less fat mass (2.22 ± 0.26 g vs 4.33 ± 0.43 g) after 13 weeks of diet, and had lower blood glucose and plasma triglycerides. There was no difference in food intake between the mice consuming quinine supplemented diet and those consuming control diet. Trpm5 knockout mice gained less fat mass than wild-type mice. There was a trend for a diet-genotype interaction for body weight and body weight gain, with the effect of quinine less pronounced in the Trpm5 KO than in the WT background. Faecal weight, energy and lipid contents were higher in quinine fed mice compared to regular AIN fed mice and in Trpm5 KO mice compared to wild type mice.
Quinine contributes to weight control in male C57BL6 mice without affecting food intake. A partial contribution of Trpm5 to quinine dependent body weight control is suggested.
Obesity; Food intake; Fat; Body composition; Gastrointestinal tract
SLC10A2-mediated reabsorption of bile acids at the distal end of the ileum is the first step in enterohepatic circulation. Because bile acids act not only as detergents but also as signaling molecules in lipid metabolism and energy production, SLC10A2 is important as the key transporter for understanding the in vivo kinetics of bile acids. SLC10A family members and the homologous genes of various species share a highly conserved region corresponding to Gly104–Pro142 of SLC10A2. The functional importance of this region has not been fully elucidated.
To elucidate the functional importance of this region, we previously performed mutational analysis of the uncharged polar residues and proline in the distal one-third (Thr130–Pro142) of the highly conserved region in mouse Slc10a2. In this study, proline and uncharged polar residues in the remaining two-thirds of this region in mouse Slc10a2 were subjected to mutational analysis, and taurocholic acid uptake and cell surface localization were examined. Cell surface localization of Slc10a2 is necessary for bile acid absorption. Mutants in which Asp or Leu were substituted for Pro107 (P107N or P107L) were abundantly expressed, but their cell surface localization was impaired. The S126A mutant was completely impaired in cellular expression. The T110A and S128A mutants exhibited remarkably enhanced membrane expression. The S112A mutant was properly expressed at the cell surface but transport activity was completely lost. Replacement of Tyr117 with various amino acids resulted in reduced transport activity. The degree of reduction roughly depended on the van der Waals volume of the side chains.
The functional importance of proline and uncharged polar residues in the highly conserved region of mouse Slc10a2 was determined. This information will contribute to the design of bile acid-conjugated prodrugs for efficient drug delivery or SLC10A2 inhibitors for hypercholesterolemia treatment.
Bile acid; Enterohepatic circulation; Ileal sodium-dependent bile acid transporter
Reactive oxygen species (ROS) are formed under natural physiological conditions and are thought to play an important role in many human diseases. A wide range of antioxidants are involved in cellular defense mechanisms against ROS, which can be generated in excess during stressful conditions, these include enzymes and non-enzymatic antioxidants. The aim of this study was to evaluate the antioxidant responses of mice to two diets control, commercial and the purified AIN 93 diet, commonly used in experiments with rodents.
Malondialdehyde (MDA) and hydrogen peroxide (H2O2) concentrations and superoxide dismutase (SOD) and glutathione reductase (GR) activities determined in the liver were lower in the group of mice fed with the AIN 93 diet, while catalase (CAT) activity was higher in the same group, when compared to the group fed on the commercial diet. Liver glutathione peroxidase (GSH-Px) activity was similar in the groups fed on either AIN 93 or the commercial diets. Two SOD isoforms, Mn-SODII and a Cu/Zn-SODV, were specifically reduced in the liver of the AIN 93 diet fed animals.
The clear differences in antioxidant responses observed in the livers of mice fed on the two diets suggest that the macro- and micro-nutrient components with antioxidant properties, including vitamin E, can promote changes in the activity of enzymes involved in the removal of the ROS generated by cell metabolism.
AIN 93 diet; Antioxidant enzymes; Commercial diet; Lipid peroxidation; Liver; Mice; Oxidative stress; Reactive oxygen species
The acute response to genotoxic carcinogens in rats is an important model for researching cancer initiation events. In this report we define the normal rat colonic epithelium by describing transcriptional events along the anterior-posterior axis and then investigate the acute effects of azoxymethane (AOM) on gene expression, with a particular emphasis on pathways associated with the maintenance of genomic integrity in the proximal and distal compartments using whole genome expression microarrays.
There are large transcriptional changes that occur in epithelial gene expression along the anterior-posterior axis of the normal healthy rat colon. AOM administration superimposes substantial changes on these basal gene expression patterns in both the distal and proximal rat colonic epithelium. In particular, the pathways associated with cell cycle and DNA damage and repair processes appear to be disrupted in favour of apoptosis.
The healthy rats’ colon exhibits extensive gene expression changes between its proximal and distal ends. The most common changes are associated with metabolism, but more subtle expression changes in genes involved in genomic homeostasis are also evident. These latter changes presumably protect and maintain a healthy colonic epithelium against incidental dietary and environmental insults. AOM induces substantial changes in gene expression, resulting in an early switch in the cell cycle process, involving p53 signalling, towards cell cycle arrest leading to the more effective process of apoptosis to counteract this genotoxic insult.
Colorectal cancer; Azoxymethane; Rats; Gene expression
Like humans, fish can be classified according to their athletic performance. Sustained exercise training of fish can improve growth and physical capacity, and recent results have documented improved disease resistance in exercised Atlantic salmon. In this study we investigated the effects of inherent swimming performance and exercise training on disease resistance in Atlantic salmon.
Atlantic salmon were first classified as either poor or good according to their swimming performance in a screening test and then exercise trained for 10 weeks using one of two constant-velocity or two interval-velocity training regimes for comparison against control trained fish (low speed continuously). Disease resistance was assessed by a viral disease challenge test (infectious pancreatic necrosis) and gene expression analyses of the host response in selected organs.
An inherently good swimming performance was associated with improved disease resistance, as good swimmers showed significantly better survival compared to poor swimmers in the viral challenge test. Differences in mortalities between poor and good swimmers were correlated with cardiac mRNA expression of virus responsive genes reflecting the infection status. Although not significant, fish trained at constant-velocity showed a trend towards higher survival than fish trained at either short or long intervals. Finally, only constant training at high intensity had a significant positive effect on fish growth compared to control trained fish.
This is the first evidence suggesting that inherent swimming performance is associated with disease resistance in fish.
This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.
In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.
To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments.
In addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na+ channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation.
For the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of neural plasticity in the pelvis and enhanced VEGF content may be associated with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain.
Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins and enables uptake of lipolysis products for energy production or storage in tissues. Our aim was to study the localization of LPL and its endothelial anchoring protein glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in mouse pancreas, and effects of diet and leptin deficiency on their expression patterns. For this, immunofluorescence microscopy was used on pancreatic tissue from C57BL/6 mouse embryos (E18), adult mice on normal or high-fat diet, and adult ob/ob-mice treated or not with leptin. The distribution of LPL and GPIHBP1 was compared to insulin, glucagon and CD31. Heparin injections were used to discriminate between intracellular and extracellular LPL.
In the exocrine pancreas LPL was found in capillaries, and was mostly co-localized with GPIHBP1. LPL was releasable by heparin, indicating localization on cell surfaces. Within the islets, most of the LPL was associated with beta cells and could not be released by heparin, indicating that the enzyme remained mostly within cells. Staining for LPL was found also in the glucagon-producing alpha cells, both in embryos (E18) and in adult mice. Only small amounts of LPL were found together with GPIHBP1 within the capillaries of islets. Neither a high fat diet nor fasting/re-feeding markedly altered the distribution pattern of LPL or GPIHBP1 in mouse pancreas. Islets from ob/ob mice appeared completely deficient of LPL in the beta cells, while LPL-staining was normal in alpha cells and in the exocrine pancreas. Leptin treatment of ob/ob mice for 12 days reversed this pattern, so that most of the islets expressed LPL in beta cells.
We conclude that both LPL and GPIHBP1 are present in mouse pancreas, and that LPL expression in beta cells is dependent on leptin.
Lipoprotein lipase; Diabetes mellitus; Islet cells; Exocrine pancreas; Endothelium; Ob/ob mice; High fat diet; Heparin; qPCR; Immunofluorescence
Lipoprotein lipase (LPL) hydrolyzes triglycerides in lipoproteins and makes fatty acids available for tissue metabolism. The activity of the enzyme is modulated in a tissue specific manner by interaction with other proteins. We have studied how feeding/fasting and some related perturbations affect the expression, in rat adipose tissue, of three such proteins, LMF1, an ER protein necessary for folding of LPL into its active dimeric form, the endogenous LPL inhibitor ANGPTL4, and GPIHBP1, that transfers LPL across the endothelium.
The system underwent moderate circadian oscillations, for LPL in phase with food intake, for ANGPTL4 and GPIHBP1 in the opposite direction. Studies with cycloheximide showed that whereas LPL protein turns over rapidly, ANGPTL4 protein turns over more slowly. Studies with the transcription blocker Actinomycin D showed that transcripts for ANGPTL4 and GPIHBP1, but not LMF1 or LPL, turn over rapidly. When food was withdrawn the expression of ANGPTL4 and GPIHBP1 increased rapidly, and LPL activity decreased. On re-feeding and after injection of insulin the expression of ANGPTL4 and GPIHBP1 decreased rapidly, and LPL activity increased. In ANGPTL4−/− mice adipose tissue LPL activity did not show these responses. In old, obese rats that showed signs of insulin resistance, the responses of ANGPTL4 and GPIHBP1 mRNA and of LPL activity were severely blunted (at 26 weeks of age) or almost abolished (at 52 weeks of age).
This study demonstrates directly that ANGPTL4 is necessary for rapid modulation of LPL activity in adipose tissue. ANGPTL4 message levels responded very rapidly to changes in the nutritional state. LPL activity always changed in the opposite direction. This did not happen in Angptl4−/− mice. GPIHBP1 message levels also changed rapidly and in the same direction as ANGPTL4, i.e. increased on fasting when LPL activity decreased. This was unexpected because GPIHBP1 is known to stabilize LPL. The plasticity of the LPL system is severely blunted or completely lost in insulin resistant rats.
Gene expression; Insulin; Gene inactivation; Cycloheximide; Actinomycin D; Transcription; Translation; Posttranslational
Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer.
Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ΔF508-CFTR (ΔF-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ΔF-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ΔF-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ΔF-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ΔF-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/ΔF heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/− heterozygotes had no difference in their responses versus +/+ wild-type mice.
Taken altogether, these data suggest that ΔF-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ΔF-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.
Cystic fibrosis (CF); CFTR; Biogenesis; CF heterozygote; Oligomer; Chloride ion channels
Regular exercises are commonly described as an important factor in health improvement, being directly related to contractile force development in cardiac cells.
In order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic Wistar rats were divided into four groups: one control (CG) and three training groups (TG’s), with low, moderate and high intensity of exercises.
In order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic Wistar rats were divided into four groups: one control (CG) and three training groups (TG’s), with low, moderate and high intensity of exercises.
Findings here reported demonstrated clear morphologic alterations, significant cellular injury and increased energy supplies at high exercise intensities. α-MyHC, as well proteins associated with mitochondrial oxidative metabolism were shown to be improved. α-MyHC expression increase 1.2 fold in high intensity training group when compared with control group. α-MyHC was also evaluated by real-time PCR showing a clear expression correlation with protein synthesis data increase in 8.48 fold in high intensity training group. Other myofibrillar protein, troponin , appear only in high intensity group, corroborating the cellular injury data. High molecular masses proteins such as MRS2 and NADH dehydrogenase, involved in metabolic pathways also demonstrate increase expression, respectily 1.5 and 1.3 fold, in response to high intensity exercise.
High intensity exercise demonstrated an increase expression in some high molecular masses myofibrilar proteins, α-MyHC and troponin. Furthermore this intensity also lead a significant increase of other high molecular masses proteins such as MRS2 and NADH dehydrogenase in comparison to low and moderate intensities. However, high intensity exercise also represented a significant degree of cellular injury, when compared with the individuals submitted to low and moderate intensities.
Heart tissue; High molecular mass proteomic; Muscle; Myofibrillar proteins; Swimming training
In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation.
A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).
We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters.
Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.
Ion; Mineral; Calcium; Transporter; Uterus; Eggshell; Chicken
Electrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments.
Intracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644).
The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.
Vascular smooth muscle; L-type Ca2+ channel; Vasoconstriction; Intracellular Ca2+; Depolarization; Window Ca2+ influx
It is well known that axotomy in the neonatal period causes massive loss of motoneurons, which is reflected in the reduction of the number of motor units and the alteration in muscle properties. This type of neuronal death is attributed to the excessive activation of the ionotropic glutamate receptors (glutamate excitotoxicity). In the present study we investigated the effect of the NMDA antagonist DAP5 [D-2-amino-5-phosphonopentanoic acid] in systemic administration, on muscle properties and on behavioural aspects following peripheral nerve injury.
Wistar rats were subjected to sciatic nerve crush on the second postnatal day. Four experimental groups were included in this study: a) controls (injection of 0.9% NaCl solution) b) crush c) DAP5 treated and d) crush and DAP5 treated. Animals were examined with isometric tension recordings of the fast extensor digitorum longus and the slow soleus muscles, as well as with locomotor tests at four time points, at P14, P21, P28 and adulthood (2 months).
1. Administration of DAP5 alone provoked no apparent adverse effects. 2. In all age groups, animals with crush developed significantly less tension than the controls in both muscles and had a worse performance in locomotor tests (p < 0.01). Crush animals injected with DAP5 were definitely improved as their tension recordings and their locomotor behaviour were significantly improved compared to axotomized ones (p < 0.01). 3. The time course of soleus contraction was not altered by axotomy and the muscle remained slow-contracting in all developmental stages in all experimental groups. EDL, on the other hand, became slower after the crush (p < 0.05). DAP5 administration restored the contraction velocity, even up to the level of control animals 4. Following crush, EDL becomes fatigue resistant after P21 (p < 0.01). Soleus, on the other hand, becomes less fatigue resistant. DAP5 restored the profile in both muscles.
Our results confirm that contractile properties and locomotor behaviour of animals are severely affected by axotomy, with a differential impact on fast contracting muscles. Administration of DAP5 reverses these devastating effects, without any observable side-effects. This agent could possibly show a therapeutic potential in other models of excitotoxic injury as well.
Observations in our laboratory provide evidence of vascular islands, defined as disconnected endothelial cell segments, in the adult microcirculation. The objective of this study was to determine if vascular islands are involved in angiogenesis during microvascular network growth.
Mesenteric tissues, which allow visualization of entire microvascular networks at a single cell level, were harvested from unstimulated adult male Wistar rats and Wistar rats 3 and 10 days post angiogenesis stimulation by mast cell degranulation with compound 48/80. Tissues were immunolabeled for PECAM and BRDU. Identification of vessel lumens via injection of FITC-dextran confirmed that endothelial cell segments were disconnected from nearby patent networks. Stimulated networks displayed increases in vascular area, length density, and capillary sprouting. On day 3, the percentage of islands with at least one BRDU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BRDU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels.
These results show that vascular islands have the ability to proliferate and suggest that they are able to incorporate into the microcirculation during the initial stages of microvascular network growth.
Angiogenesis; Microcirculation; Mesentery; Proliferation; Endothelial cell
Data on blood flow regulation, renal filtration, and urine output in salt-sensitive Dahl S rats fed on high-salt (hypertensive) and low-salt (prehypertensive) diets and salt-resistant Dahl R rats fed on high-salt diets were analyzed using a mathematical model of renal blood flow regulation, glomerular filtration, and solute transport in a nephron.
The mechanism of pressure-diuresis and pressure-natriuresis that emerges from simulation of the integrated systems is that relatively small increases in glomerular filtration that follow from increases in renal arterial pressure cause relatively large increases in urine and sodium output. Furthermore, analysis reveals the minimal differences between the experimental cases necessary to explain the observed data. It is determined that differences in renal afferent and efferent arterial resistances are able to explain all of the qualitative differences in observed flows, filtration rates, and glomerular pressure as well as the differences in the pressure-natriuresis and pressure-diuresis relationships in the three groups. The model is able to satisfactorily explain data from all three groups without varying parameters associated with glomerular filtration or solute transport in the nephron component of the model.
Thus the differences between the experimental groups are explained solely in terms of difference in blood flow regulation. This finding is consistent with the hypothesis that, if a shift in the pressure-natriuresis relationship is the primary cause of elevated arterial pressure in the Dahl S rat, then alternation in how renal afferent and efferent arterial resistances are regulated represents the primary cause of chronic hypertension in the Dahl S rat.
The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR).
Our aim was to analyze and compare angiogenic factor expression levels in both subcutaneous (SC) and omentum (OM) adipose tissues from morbidly obese patients (n = 26) with low (OB/L-IR) (healthy obese) and high (OB/H-IR) degrees of IR, and lean controls (n = 17). Another objective was to examine angiogenic factor correlations with obesity and IR.
Here we found that VEGF-A was the isoform with higher expression in both OM and SC adipose tissues, and was up-regulated 3-fold, together with MMP9 in OB/L-IR as compared to leans. This up-regulation decreased by 23% in OB/-H-IR compared to OB/L-IR. On the contrary, VEGF-B, VEGF-C and VEGF-D, together with MMP15 was down-regulated in both OB/H-IR and OB/L-IR compared to lean patients. Moreover, MMP9 correlated positively and VEGF-C, VEGF-D and MMP15 correlated negatively with HOMA-IR, in both SC and OM.
We hereby propose that the alteration in MMP15, VEGF-B, VEGF-C and VEGF-D gene expression may be caused by one of the relevant adipose tissue processes related to the development of IR, and the up-regulation of VEGF-A in adipose tissue could have a relationship with the prevention of this pathology.
Vascular Endothelial Growth Factor and Metalloproteinase; Obesity; Insulin Resistance; Omentum Adipose Tissue; Subcutaneous Adipose Tissue
Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.
Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.
The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
Heart; Screening; Zebrafish; Small molecule; Ex vivo; Ca2+ signaling
Matrix metalloproteinases (MMPs) 2 and 9 are two gelatinase members which have been found elevated in exudative pleural effusions. In endothelial cells these MMPs increase paracellular permeability via the disruption of tight junction (TJ) proteins occludin and claudin. In the present study it was investigated if MMP2 and MMP9 alter permeability properties of the pleura tissue by degradation of TJ proteins in pleural mesothelium.
In the present study the transmesothelial resistance (RTM) of sheep pleura tissue was recorded in Ussing chambers after the addition of MMP2 or MMP9. Both enzymes reduced RTM of the pleura, implying an increase in pleural permeability. The localization and expression of TJ proteins, occludin and claudin-1, were assessed after incubation with MMPs by indirect immunofluorescence and western blot analysis. Our results revealed that incubation with MMPs did not alter neither proteins localization at cell periphery nor their expression.
MMP2 and MMP9 increase the permeability of sheep pleura and this finding suggests a role for MMPs in pleural fluid formation. Tight junction proteins remain intact after incubation with MMPs, contrary to previous studies which have shown TJ degradation by MMPs. Probably MMP2 and MMP9 augment pleural permeability via other mechanisms.
The transcription factor c-myc regulates genes involved in hepatocyte growth, proliferation, metabolism, and differentiation. It has also been assigned roles in liver development and regeneration. In previous studies, we made the unexpected observation that c-Myc protein levels were similar in proliferating fetal liver and quiescent adult liver with c-Myc displaying nucleolar localization in the latter. In order to investigate the functional role of c-Myc in adult liver, we have developed a hepatocyte-specific c-myc knockout mouse, c-mycfl/fl;Alb-Cre.
Liver weight to body weight ratios were similar in control and c-myc deficient mice. Liver architecture was unaffected. Conditional c-myc deletion did not result in compensatory induction of other myc family members or in c-Myc's binding partner Max. Floxed c-myc did have a negative effect on Alb-Cre expression at 4 weeks of age. To explore this relationship further, we used the Rosa26 reporter line to assay Cre activity in the c-myc floxed mice. No significant difference in Alb-Cre activity was found between control and c-mycfl/fl mice. c-myc deficient mice were studied in a nonproliferative model of liver growth, fasting for 48 hr followed by a 24 hr refeeding period. Fasting resulted in a decrease in liver mass and liver protein, both of which recovered upon 24 h of refeeding in the c-mycfl/fl;Alb-Cre animals. There was also no effect of reducing c-myc on recovery of liver mass following 2/3 partial hepatectomy.
c-Myc appears to be dispensable for normal liver growth during the postnatal period, restoration of liver mass following partial hepatectomy and recovery from fasting.
Sedentary lifestyle is associated with coronary artery disease but even shorter periods of physical inactivity may increase cardiovascular risk. Cystatin C is independently associated with cardiovascular disease and our objective was to investigate the relation between this novel biomarker and standardized bed rest. Research of immobilization physiology in humans is challenging because good biological models are in short supply. From the Women International Space simulation for Exploration study (WISE) we studied markers of atherosclerosis and kidney function, including cystatin C, in a standardized bed rest study on healthy volunteers. Fifteen healthy female volunteers participated in a 20-day ambulatory control period followed by 60 days of bed rest in head-down tilt position (-6°) 24 h a day, finalized by 20 days of recovery. The subjects were randomized into two groups during bed rest: a control group (n = 8) that remained physically inactive and an exercise group (n = 7) that participated in both supine resistance and aerobic exercise training.
Compared to baseline values there was a statistically significant increase in cystatin C in both groups after bed rest (P < 0.001). Glomerular filtration rate (GFR), calculated by both cystatin C and Cockcroft-Gault equation, decreased after bed rest while there were no differences in creatinine or creatine kinase levels. CRP did not change during bed rest in the exercise group, but there was an increase of CRP in the control group during recovery compared to both the baseline and the bed rest periods. The apo-B/apo-Ai ratio increased during bed rest and decreased again in the recovery period. Subjects experienced a small but statistically significant reduction in weight during bed rest and compared to baseline weights remained lower at day 8 of recovery.
During and following prolonged standardized bed rest the concentrations of several clinically relevant cardiovascular risk markers change.
This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity.
Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder.
The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.
NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density.
For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.
Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery.
Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times.
This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.
Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs.
The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established.
From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.
Bears are among the most physiologically remarkable mammals. They spend half their life in an active state and the other half in a state of dormancy without food or water, and without urinating, defecating, or physical activity, yet can rouse and defend themselves when disturbed. Although important data have been obtained in both captive and wild bears, long-term physiological monitoring of bears has not been possible until the recent advancement of implantable devices.
Insertable cardiac monitors that were developed for use in human heart patients (Reveal® XT, Medtronic, Inc) were implanted in 15 hibernating bears. Data were recovered from 8, including 2 that were legally shot by hunters. Devices recorded low heart rates (pauses of over 14 seconds) and low respiration rates (1.5 breaths/min) during hibernation, dramatic respiratory sinus arrhythmias in the fall and winter months, and elevated heart rates in summer (up to 214 beats/min (bpm)) and during interactions with hunters (exceeding 250 bpm). The devices documented the first and last day of denning, a period of quiescence in two parturient females after birthing, and extraordinary variation in the amount of activity/day, ranging from 0 (winter) to 1084 minutes (summer). Data showed a transition toward greater nocturnal activity in the fall, preceding hibernation. The data-loggers also provided evidence of the physiological and behavioral responses of bears to our den visits to retrieve the data.
Annual variations in heart rate and activity have been documented for the first time in wild black bears. This technique has broad applications to wildlife management and physiological research, enabling the impact of environmental stressors from humans, changing seasons, climate change, social interactions and predation to be directly monitored over multiple years.
Electrophysiology; Hibernation; Cardiac Physiology
In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells.
mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production.
The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2.
These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.