Effect of feeding schedule on apparent energy and amino acid digestibility by growing pigs

An experiment was conducted to investigate the effects of feeding schedule on dry matter (DM), gross energy (GE), crude protein (CP), and amino acid (AA) digestibility by growing pigs. Six growing castrates (initial body weight: 40.5 ± 2.25 kg) were equipped with a T-cannula in the distal ileum and allotted to a repeated 3 × 3 Latin square design with three animals and three periods in each square. A maize–soybean meal based diet (181 g CP/kg) was formulated and fed to the pigs in three different feeding schedules; i.e., one meal per day, two meals per day, or free access to feed. Pigs fed one or two meals per day were allotted a daily amount of feed equivalent to three times the maintenance energy requirement. Fecal and ileal samples were collected from all pigs in each period. The coefficients of ileal apparent digestibility (CIAD) for DM, GE, CP, and AA were calculated as were the coefficients of total tract apparent digestibility (CTTAD) of DM and GE. Results of the experiment showed that there were no differences in the CIAD for DM, GE, or for any of the AA among the three feeding schedules. In contrast, pigs given free access to the diet had lower (P<0.002) CTTAD for DM and GE compared with pigs fed a restricted amount of feed either once or twice daily. Therefore, the concentration of digestible energy in the diet was lower if calculated from pigs given free access to the diet compared with pigs fed the diet once or twice daily (14.38 MJ/kg versus 14.83 MJ/kg and 15.13 MJ/kg). It is concluded that the feeding schedule does not influence the CIAD for DM, GE, CP, or AA by pigs. Therefore, any of the three feeding schedules that were used in the current experiment may be used in experiments aimed at measuring CIAD in pigs. However, if the CTTAD of DM and GE are measured, it is recommended that pigs are given free access to feed.     

Oral cancer,cigarette smoke and mitochondrial 18 kDa translocator protein (TSPO) — In vitro,in vivo,salivary analysis

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Δψ_m), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p < 0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133 ± 643 fmol/mg protein and 6956 ± 549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p < 0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p < 0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p < 0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p < 0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.     

Influence of diet complexity on productive performance and nutrient digestibility of weanling pigs

The effect of diet complexity on coefficient of total tract apparent digestibility (CTTAD) and growth performance was studied in piglets from 21 to 62 d of age. There were five experimental prestarter diets (21–41 d of age) with similar net energy and total indispensable amino acids content. The negative control diet contained 400 g raw maize, 40 g fish meal (FM) and 70 g lactose (LAC)/kg and the positive control diet contained 400 g cooked maize, 100 g FM and 140 g LAC/kg. The other three diets were similar to the positive control diet but the cooked maize was substituted by raw maize or contained 40 g FM/kg or 70 g LAC/kg, respectively. Each treatment was replicated six times (six pigs per pen). For the starter period (42–62 d of age), half of the pens of each of the prestarter treatments was sorted into two groups and fed either a standard soybean meal–raw maize–lard diet or a diet with similar nutrient profile that included 200 g cooked maize, 50 g FM, 13 g LAC, 20 g soy protein concentrate and 10 g soybean oil/kg in substitution of lower cost ingredients. Dietary treatment did not affect piglet performance at any age, but incidence of diarrhoea during the prestarter period, was higher in piglets fed the negative control diet than in piglets fed any of the other diets (P<0.05). At 30 d of age (prestarter period), the CTTAD of organic matter and gross energy were lower (P<0.001) for pigs fed the negative control diet than for pigs fed the other diets, but that of crude protein was not affected. At 50 d of age (starter period), dietary treatment did not affect the CTTAD of any dietary component. It is concluded that the use of high levels of high quality ingredients in the diet did not improve growth performance of piglets at any age. From 21 to 41 d of age, the incidence of diarrhoea was reduced and the CTTAD of dietary components was increased when the more complex diets were fed. The inclusion of high levels of high quality ingredients in the diet to maximize performance of young pigs might not be justified under all circumstances.     

Two populations of TSPO binding sites in oral cancer SCC-15 cells

Oral cancer mortality and morbidity rates remain high. The main inducer of oral cancer is cigarette smoke (CS). Translocator protein 18 kDa (TSPO) was shown to play a role in carcinogenesis. We characterized TSPO binding sites in human oral cancer cell line SCC-15 and examined effect of CS on TSPO binding. We exposed SCC-15 human squamous cells to cigarette smoke. [³H]PK 11195 binding results were assessed in cells confluent for one day. To characterize the number of population sites, a custom written Matlab program compared Pearson linear correlation coefficients between all points in the Scatchard plot. Using [³H]PK 11195 as a radio ligand, we found that TSPO binding sites are not uniform, but separated into two sub-populations, one with high affinity (respective Kd and Bmax values of 1.40±0.08 nM and 1586±48 fmol/mg protein), another with lower affinity (respective Kd and Bmax values of 61±5 nM and 26260±1050 fmol/mg protein). We demonstrate rapid decrease in TSPO binding to the high affinity site induced by exposure to CS; specifically, significant 36% decrease in binding after 30 min CS exposure (p<0.05), and 69% decrease after 2 h CS exposure (p<0.05). Association between TSPO and CS exposure may contribute to understanding the underlying mechanism of oral carcinogenesis.     

Sphingomyelin synthase 2 over-expression induces expression of aortic inflammatory biomarkers and decreases circulating EPCs in ApoE KO mice

AimsThis study sought to assess the effect of sphingomyelin synthase 2 (SMS2) over-expression on plaque component and endothelial dysfunction in atherosclerosis.Main methodsWe generated recombinant adenovirus vectors containing human SMS2 cDNA (AdV-SMS2) or control gene GFP cDNA (AdV-GFP). Both AdVs were injected (i.v.) into ApoE KO mice to establish SMS2 over-expressing and control mice models, respectively. The mice were fed a high fat diet for 30 days. We then examined their plasma lipid levels, expression levels of aortic inflammatory biomarkers critical for the plaque's stability, and numbers of peripheral endothelial progenitor cells (EPC).Key findingsCompared with the control mice, SMS2 over-expression had significantly (1) increased aortic matrix metalloproteinase-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), tissue factor (TF) and cyclooxygenase-2 (COX-2) mRNA levels (1.9-fold, 2.2-fold, 2.6-fold and 3.2-fold, respectively, P < 0.01) and protein levels (2.2-fold, 1.9-fold, 1.9-fold and 2.1-fold, respectively, P < 0.01); (2) increased MMP-2, COX-2 in situ expression in aortic root (2.6-fold and 2.3-fold, respectively, P < 0.01); (3) decreased aortic COX-1 mRNA levels (65%, P < 0.01) and protein levels (64%, P < 0.01); and (4) decreased CD34/KDR-positive cells (33%, P < 0.01), circulating angiogenic cells (CACs) (50%, P < 0.05), and colony forming units (CFUs) (40%, P < 0.05) in circulation.SignificanceSMS2 over-expression was probably associated with increased expression of aortic inflammatory biomarkers, as well as decreased numbers of CD34/KDR-positive cells, CACs and CFUs in circulation. Therefore, SMS2 over-expression might correlate with endothelial dysfunction and aggravate atherosclerotic plaque instability in ApoE KO mice.     

Lipoic acid effects on established atherosclerosis

AimsAlpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis.Main methodsWatanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20 mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-κB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA).Key findingsLA decreased body weight by 15 ± 5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-κB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy.SignificanceThe present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.     

Investigating the interactions of the 18 kDa translocator protein and its ligand PK11195 in planar lipid bilayers

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18 kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~ 10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51 Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug–membrane interactions may contribute to the apparent affinity of TSPO ligands.     

Pectin and psyllium decrease the susceptibility of LDL to oxidation in guinea pigs

These studies were undertaken to determine whether pectin (PE) and psyllium (PSY) intake affect the circulating levels of alpha-tocopherol and the susceptibility of low density lipoprotein (LDL) to oxidation. For that purpose, male Hartley guinea pigs were fed 19 g/100 g of a fat mix with a 2:1:1 ratio of saturated:polyunsaturated:monounsaturated fatty acids and 35 g/100 g total carbohydrate with 80% of the carbohydrate energy contributed by sucrose. Diets were identical in composition except for the fiber source: cellulose (control diet), PE, or PSY. Guinea pigs fed PE or PSY had 36% and 67% lower plasma cholesterol concentrations, respectively, compared with controls (P < 0.001). This plasma cholesterol lowering was associated with both very low density lipoproteins and LDL cholesterol fractions. Intake of PE or PSY resulted in 54% lower plasma triacylglycerol (TAG) concentrations compared with the control group (P < 0.001). LDL from PE and PSY fed guinea pigs contained fewer molecules of cholesteryl ester, and alpha-tocopherol concentrations in this particle were 49% and 66% higher, respectively, compared with controls. In addition, LDL from guinea pigs fed soluble fiber exhibited less susceptibility to oxidation than those from the control group, as determined by thiobarbituric acid-reactive substances formation. Hepatic free and esterified cholesterol were 32% lower and hepatic TAG was 25% lower in guinea pigs fed PE and PSY compared with controls. The data from these studies confirm that PE and PSY reverse the hyperlipidemia associated with high fat-sucrose diets and demonstrate a potential antioxidant effect of soluble fiber on circulating LDL.     

Accumulation of copper in the kidney of pigs fed high dietary zinc is due to metallothionein expression with minor effects on genes involved in copper metabolism

A study was conducted to determine the effect of high dietary zinc (Zn) oxide on trace element accumulation in various organs with special emphasis on the kidney. A total of 40 weaned piglets were allocated into two groups with 16 and 24 piglets each receiving a diet containing normal (NZn; 100 mg Zn/kg) or high (HZn; 2,100 mg Zn/kg) Zn concentration, respectively. After two weeks, eight piglets from each treatment were killed and organ samples were taken. Eight piglets from the remaining 16 pigs fed HZn diets were changed to NZn diets (CZn). All remaining piglets were killed after another two weeks for organ sampling. Trace element concentration was determined in the jejunum, liver, kidney, pancreas, bone (metacarpal IV), spleen, lung, thymus, tonsils and lymph nodes of jejunum, ileum and colon. Kidney mRNA expression of Zn transporter ZnT1 and ZIP4, genes involved in Cu metabolism (Ctr1, Atox1, SOD1, ATP7A, CCS, CP) and divalent metal ion transport (DMT1) and binding (MT-1a, MT-2b, MT-3) were determined. The Zn concentration in jejunum, liver, pancreas tissue and metacarpal IV was higher (P < 0.05) in HZn group compared with NZn and CZn groups. Trace element concentration in organs of CZn pigs was similar to those fed NZn diets. Zn concentration in muscle, lung and lymphatic organs as thymus, tonsils, spleen and lymph nodes of jejunum, ileum and colon did not differ between the groups. Zn and Cu were positively correlated (R = 0.67; P < 0.05) in the kidney. No significant differences for Cu chaperones, Cu transporters and Cu-dependent factors were determined despite decreased expression of Atox1 after two weeks and increased Ctr1 expression over time in the HZn group. Expression of MT-1a, MT-2b and MT-3 were significantly higher in HZn fed pigs with most pronounced effects for MT-1a > MT-2b > MT-3. Gene expression of MTs in pigs fed CZn diets did not differ from pigs fed NZn diets. The data suggest that high dietary Zn feeding in pigs leads to Cu co-accumulation in the kidney of pigs with minor effect on genes relevant for Cu metabolism. In addition, the organ Zn and Cu accumulation is reversible after two weeks of withdrawal of high dietary Zn.     

The alkaloid matrine of the root of Sophora flavescens prevents arrhythmogenic effect of ouabain

Matrine, a alkaloid of the root of Sophora flavescens, has multiple protective effects on the cardiovascular system including cardiac arrhythmias. However, the molecular and ionic mechanisms of matrine have not been well investigated. Our study aimed at to shed a light on the issue to investigate the antiarrhythmic effects of matrine by using ouabain to construct an arrhythmic model of cardiomyocytes. In this experiment, matrine significantly and dose-dependently increased the doses of ouabain required to induce cardiac arrhythmias and decreased the duration of arrhythmias in guinea pigs. In cardiomyocytes of guinea pigs, ouabain 10 μM prolonged action potential duration by 80% (p < 0.05) and increased L-type Ca²⁺ currents and Ca²⁺ transients induced by KCl (p < 0.05). Matrine 100 μM shortened the prolongation of APD and prevented the increase of L-type Ca²⁺ currents and Ca²⁺ transients induced by ouabain. Taken together, these findings provide the first evidence that matrine possessed arrhythmogenic effect of ouabain by inhibiting of L-type Ca²⁺ currents and Ca²⁺ overload in guinea pigs.     

Construction and validation of an atomic model for bacterial TSPO from electron microscopy density,evolutionary constraints,and biochemical and biophysical data

The 18 kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications.     

Adipose tissue deficiency results in severe hyperlipidemia and atherosclerosis in the low-density lipoprotein receptor knockout mice

Adipose tissue can store over 50% of whole-body cholesterol; however, the physiological role of adipose tissue in cholesterol metabolism and atherogenesis has not been directly assessed. Here, we examined lipoprotein metabolism and atherogenesis in a unique mouse model of severe lipodystrophy: the Seipin^−/− mice, and also in mice deficient in both low-density lipoprotein receptor (Ldlr) and Seipin: the Ldlr^−/− Seipin^−/− mice. Plasma cholesterol was moderately increased in the Seipin^−/− mice when fed an atherogenic diet. Strikingly, plasma cholesterol reached ~ 6000 mg/dl in the Seipin^−/− Ldlr^−/− mice on an atherogenic diet, as compared to ~ 1000 mg/dl in the Ldlr^−/− mice on the same diet. The Seipin^−/− Ldlr^−/− mice also developed spontaneous atherosclerosis on chow diet and severe atherosclerosis on an atherogenic diet. Rosiglitazone treatment significantly reduced the hypercholesterolemia of the Seipin^−/− Ldlr^−/− mice, and also alleviated the severity of atherosclerosis. Our results provide direct evidence, for the first time, that the adipose tissue plays a critical role in the clearance of plasma cholesterol. Our results also reveal a previously unappreciated strong link between adipose tissue and LDLR in plasma cholesterol metabolism.     

Heterogeneous infectiousness in guinea pigs experimentally infected with Trypanosoma cruzi

Guinea pigs are important reservoirs of Trypanosoma cruzi, the causative parasite of Chagas disease, and in the Southern Cone of South America, transmission is mediated mainly by the vector Triatoma infestans. Interestingly, colonies of Triatoma infestans captured from guinea pig corrals sporadically have infection prevalence rates above 80%. Such high values are not consistent with the relatively short 7–8 week parasitemic period that has been reported for guinea pigs in the literature. We experimentally measured the infectious periods of a group of T. cruzi-infected guinea pigs by performing xenodiagnosis and direct microscopy each week for one year. Another group of infected guinea pigs received only direct microscopy to control for the effect that inoculation by triatomine saliva may have on parasitemia in the host. We observed infectious periods longer than those previously reported in a number of guinea pigs from both the xenodiagnosis and control groups. While some guinea pigs were infectious for a short time, other “super-shedders” were parasitemic up to 22 weeks after infection, and/or positive by xenodiagnosis for a year after infection. This heterogeneity in infectiousness has strong implications for T. cruzi transmission dynamics and control, as super-shedder guinea pigs may play a disproportionate role in pathogen spread.     

Methane output of rabbits (Oryctolagus cuniculus) and guinea pigs (Cavia porcellus) fed a hay-only diet: implications for the scaling of methane production with body mass in non-ruminant mammalian herbivores

It is assumed that small herbivores produce negligible amounts of methane, but it is unclear whether this is a physiological peculiarity or simply a scaling effect. A respiratory chamber experiment was conducted with six rabbits (Oryctolagus cuniculus, 1.57 ± 0.31 kg body mass) and six guinea pigs (Cavia porcellus, 0.79 ± 0.07 kg) offered grass hay ad libitum. Daily dry matter (DM) intake and DM digestibility were 50 ± 6 g kg^? 0.75 d^? 1 and 55 ± 6% in rabbits and 59 ± 11 g kg^? 0.75 d^? 1 and 61 ± 3% in guinea pigs, respectively. Methane production was similar for both species (0.20 ± 0.10 L d^? 1 and 0.22 ± 0.08 L d^? 1) and represented 0.69 ± 0.32 and 1.03 ± 0.29% of gross energy intake in rabbits and guinea pigs, respectively. In relation to body mass (BM) guinea pigs produced significantly more methane. The data on methane per unit of BM obtained in this study and from the literature on the methane output of elephant, wallabies and hyraxes all lay close to a regression line derived from roughage-fed horses, showing an increase in methane output with BM. The regression, including all data, was nearly identical to that based on the horse data only (methane production in horses [L d^? 1] = 0.18 BM [kg]^{0.97 (95%CI 0.92–1.02)}) and indicates linear scaling. Because feed intake typically scales to BM^0.75, linear scaling of methane output translates into increasing energetic losses at increasing BM. Accordingly, the data collection indicates that an increasing proportion of ingested gross energy is lost because relative methane production increases with BM. Different from ruminants, such losses (1%–2% of gross energy) appear too small in non-ruminant herbivores to represent a physiologic constraint on body size. Nevertheless, this relationship may represent a physiological disadvantage with increasing herbivore body size.     

Effects of feeding high- and low-fibre fractions of air-classified,solvent-extracted canola meal on diet nutrient digestibility and growth performance of weaned pigs

The dietary energy value of solvent-extracted canola meal (CM) is limited by its relative high fibre content. The fibre-rich hull of canola is denser than the oil-free cotyledons, so these seed components partially fractionate in a stream of air. Air classification thus separates CM into a low-fibre, light-particle fraction and a high-fibre, heavy-particle fraction of interest for feeding monogastric and ruminant species, respectively. Crude fibre (CF), acid detergent fibre (ADF) and neutral detergent fibre (NDF) in light-particle fraction were reduced by 96, 34 and 28% compared with CM (83 CF, 165 ADF, 238 NDF g/kg, as-is). Brassica (B) napus, Brassica juncea, or their fractions were evaluated feeding 288 weaned pigs (7.1 kg) for 37 d as a 2 × 3 factorial with 12 replicate pens per treatment. Wheat-based diets including 200 g of test feedstuff/kg provided 10.5 and 10.0 MJ net energy (NE)/kg and 1.27 and 1.15 g standardised ileal digestible lysine/MJ NE and were fed for 9 and 28 d, respectively. Pen feed added, orts, and individual pig body weight were measured weekly to calculate average daily feed intake (ADFI), average daily gain (ADG), and feed efficiency (G:F). Pen faecal samples were collected on d 16 and 17 to calculate diet apparent total tract digestibility coefficients (CATTD) of dry matter (DM), gross energy (GE), crude protein (CP) and digestible energy (DE) value. Pigs fed B. juncea had 3 and 2% higher (P<0.001) CATTD of DM (0.82 vs. 0.79) and GE (0.84 vs. 0.82) than pigs fed B. napus. Feeding the light-particle fraction increased (P<0.001) CATTD of DM (0.82 vs. 0.79), GE (0.84 vs. 0.82), and CP (0.79 vs. 0.77) by 4, 3 and 3% compared with CM, respectively. For the entire trial, pigs fed B. juncea consumed 33 g/d less (P<0.001) feed (723 vs. 756 g/d), had 0.02 higher (P<0.05) G:F (0.735 vs. 0.718 g:g), but ADG (503 vs. 514 g/d) was not different (P>0.05) compared to pigs fed B. napus. Feeding pigs the light-particle fractions did not affect (P>0.05) ADFI (741 vs. 736 g/d), increased (P<0.05) G:F 0.02 (0.739 vs. 0.721 g:g) and tended to increase (P=0.07) ADG (519 vs. 501 g/d) by 18 g/d compared to CM. In conclusion, air classification of canola meal increased diet nutrient digestibility, but only modestly increased G:F of weaned pigs due to dietary fibre reduction.     

Febrile and cortisol responses induced in guinea pigs by localized peripheral inflammatory stimulation

(1) In guinea pigs, a high (100 μg/kg) or a low (10 μg/kg) dose of lipopolysaccharide (LPS) was injected into subcutaneously implanted Teflon chambers along with the prior injection of a local anesthetic (ropivacaine) or sterile saline. (2) Intra-chamber injection of the LPS alone induced fever and elevation of circulating cortisol. (3) Fever in response to the low dose of the LPS was attenuated by the pretreatment with the local anesthetic while circulating levels of cortisol were not impaired by this procedure. (4) There was a moderate increase in plasma interleukin-6 (IL-6) in response to both concentrations of locally administered LPS. The LPS did not enter the systemic circulation in measurable amounts. (5) These results favor the possibility of a participation of afferent neural as well as humoral signals (IL-6) in the manifestation of fever in this experimental model.     

Novel androstenetriol interacts with the mitochondrial translocator protein and controls steroidogenesis

Steroid hormones are metabolically derived from multiple enzymatic transformations of cholesterol. The controlling step in steroid hormone biogenesis is the delivery of cholesterol from intracellular stores to the cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator protein (TSPO) plays an integral part in this mitochondrial cholesterol transport. Consistent with its role in intracellular cholesterol movement, TSPO possesses a cholesterol recognition/interaction amino acid consensus (CRAC) motif that has been demonstrated to bind cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig tumor cells and constitutive steroidogenesis in R2C rat Leydig tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the steroidogenic acute regulatory protein without any effect on TSPO protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive steroid formation.     

The role of insulin growth factor on atherosclerosis and endothelial function: the effect on hyperlipidemia and aging

AimsInsulin/insulin-like growth factor (IGF-1) signaling is important for a variety of age-related processes. However, whether or not it affects atherosclerosis is unknown.Main methodsSix groups of 6 male New Zealand white rabbits were treated for 12 weeks under the following conditions: Groups YC and YIGF: Young rabbits (10 weeks old) were fed regular chow w/wo IGF-1(Somazon0.1 mg/kg/day, s.c.). Groups HC and HIGF: young rabbits were fed HCD (0.5% cholesterol plus regular chow) w/wo IGF-1. Groups OC and OIGF: old rabbits (120 weeks old) were fed regular chow w/wo IGF-1.Key findingsPlasma lipid levels, endothelial responses and morphological findings did not differ between groups YIGF and YC. Animals in group HC had increased plasma lipid levels and atheromas. In group HIGF, IGF led to atheromas with increased plasma insulin growth factor binding protein 3 (IBP3), inducible nitric oxide synthase(iNOS) expression and nitrotyrosine staining, macrophage staining, SM1 staining and SM embryo staining compared to HC. Basal nitric oxide (NO) release evaluated by plasma NO metabolites (NOx) and cGMP levels were lowest in the HIGF group.SignificanceOverall, IGF-1 promoted atherosclerosis by affecting endothelial function and aging. These findings indicate that Insulin/IGF1 may contribute to atherogenesis in the elderly.     

Development and validation of a sensitive HPLC method for the quantification of HI-6 in guinea pig plasma and evaluated in domestic swine

A rapid and small volume assay to quantify HI-6 in plasma was developed to further the development and licensing of an intravenous formulation of HI-6. The objective of this method was to develop a sensitive and rapid assay that clearly resolved HI-6 and an internal standard in saline and plasma matrices. A fully validated method using ion-pair HPLC and 2-PAM as the internal standard fulfilled these requirements. Small plasma samples of 35 μL were extracted using acidification, filtration and neutralization. Linearity was shown for over 4 μg/mL to 1 mg/mL with accuracy and precision within 6% relative error at the lower limit of detection. This method was utilized in the pharmacokinetic analysis HI-6 dichloride (2Cl) and HI-6 dimethane sulfonate (DMS) in anaesthetized guinea pigs and domestic swine following an intravenous bolus administration. From the resultant pharmacokinetic parameters a target plasma concentration of 100 μM was established and maintained in guinea pigs receiving an intravenous infusion. This validated method allows for the analysis of low volume samples, increased sample numbers and is applicable to the determination of pharmacokinetic profiles and parameters.