Concentrations In Peripheral Circulation Research Articles

Fructose Reduces Mitochondrial Metabolism and Increases Extracellular BCAA during Insulin Resistance in C2C12 Myotubes.

C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.

Secretory leucocyte protease inhibitor (SLPI) as an adjunct prognostic biomarker for canine pyometra.

Pyometra is a potentially life-threatening condition that affects intact female dogs in their middle to advance age. Timely diagnosis and appropriate treatment are critical for the survival of patients, especially when pyometra advances to sepsis. This study aimed to investigate the prognostic potential of certain haematology, serum biochemical and inflammatory biomarker, secretory leucocyte protease inhibitor (SLPI) for pyometra in bitches (n = 41). Blood samples were collected after clinical diagnosis of pyometra for haematology and serum biochemistry. Based on the prognosis following medical/surgical treatment, animals were retrospectively categorized into survivor (n = 29) and dead (n = 12). Endometrial tissue sections were obtained from the bitches undergoing ovariohysterectomy (n = 21). Serum concentration of SLPI was quantified using sandwich ELISA and its expression in the endometrium was investigated using RT-qPCR. A marked increase in the total leucocyte count (TLC), neutrophils, blood urea nitrogen (BUN) and serum creatinine was observed in the female dogs that did not survive. Significant elevation in the serum SLPI concentration (3.49 ± 0.44 vs. 2.38 ± 0.13 ng/mL) was observed in the bitches those died after the treatment, in comparison to those survived (p < .01). Additionally, there was a notable upregulation of SLPI in the endometrium in the bitches those died due to pyometra. Based on the ROC analysis results, it was observed that a cut-off concentration of 2.93 ng/mL for SLPI, 27.77 mg/dL for BUN and 16.3 × 103 /μL for TLC could effectively distinguish the prognosis of pyometra-affected dogs. From this study, it can be concluded that upregulation of SLPI in the endometrium and its elevated concentration in peripheral circulation along with TLC and BUN concentration could serve as valuable indicators for predicting the prognosis of pyometra in bitches.

Glucagon regulates lipolysis and fatty acid oxidation through inositol triphosphate receptor1 in the liver.

Glucagon promotes hepatic glucose production, lipolysis and amino acid catabolism. Inhibition of glucagon activity, or glucagon resistance, not only lowers blood glucose levels, but also induces non-alcoholic fatty liver disease and hyperaminoacidemia.

Pregnancy-associated glycoprotein profile in milk and its relationship with the circulating level during early pregnancy in goats

The level of pregnancy-associated glycoprotein (PAG) in milk and its relationship with the concentration in peripheral circulation during early pregnancy remain uncertain in goats. Therefore, the objectives of present study were to investigate temporal profile of PAG in milk (whole and skim milk) and blood, and to determine the relationship between milk and circulating PAG level during early pregnancy in goats. Milk and plasma samples were collected from multiparous goats (n = 5) on alternate day from d 26 to 30, and thereafter weekly till d 51 of gestation to estimate PAG level using commercial ELISA tests. Pregnancies were confirmed at d 37 of gestation by transrectal ultrasonography (USG). The eff ;ects of days of pregnancy and animal status (pregnant vs. non-pregnant) on PAG levels in milk and plasma were analyzed using linear mixed model. Compared to USG, assay accuracy were 94.4%, 88.9% and 93.3% for whole milk, skim milk and plasma ELISA tests, respectively. Maternal PAG level in milk (both whole and skim milk) and circulation progressively increased with the days of pregnancy during early gestation (P < 0.001). On the contrary, the PAG level of corresponding milk and plasma samples of non-pregnant goats remain unchanged (P > 0.05) and below to cut-off values throughout the experiment. PAG levels in whole and skim milk samples of pregnant goats were similar (P > 0.05) at all-time points with approximately 2-folds lower PAG compared to the plasma samples. A strong positive correlation and high agreement of plasma PAG with PAG levels in whole and skim milk were detected. By using milk, the ELISA showed sensitivity of 100% after d 28 of gestation. In conclusion, the PAG level in milk and peripheral circulation is affected by the day of gestation during early pregnancy and the optimal time to carry out first pregnancy diagnosis using milk or plasma based ELISA is on and after d 28 of gestation. The strong relationship and high agreement of milk PAG with its level in peripheral circulation supports that milk (both whole and skim milk) is a good alternative to plasma for early pregnancy diagnosis from d 28 to d 51 and can effectively be used as a marker of placental functions and fetal health in goats.

Norepinephrine stimulates mobilization of endothelial progenitor cells after limb ischemia.

ObjectiveDuring several pathological processes such as cancer progression, thermal injury, wound healing and hindlimb ischemia, the mobilization of endothelial progenitor cells (EPCs) mobilization was enhanced with an increase of sympathetic nerve activity and norepinephrine (NE) secretion, yet the cellular and molecular mechanisms involved in the effects of NE on EPCs has less been investigated.Methods and ResultsEPCs from BMs, peripheral circulation and spleens, the VEGF concentration in BM, skeletal muscle, peripheral circulation and spleen and angiogenesis in ischemic gastrocnemius were quantified in mice with hindlimbs ischemia. Systemic treatment of NE significantly increased EPCs number in BM, peripheral circulation and spleen, VEGF concentration in BM and skeletal muscle and angiogenesis in ischemic gastrocnemius in mice with hind limb ischemia, but did not affair VEGF concentration in peripheral circulation and spleen. EPCs isolated from healthy adults were cultured with NE in vitro to evaluate proliferation potential, migration capacity and phosphorylations of Akt and eNOS signal moleculars. Treatment of NE induced a significant increase in number of EPCs in the S-phase in a dose-dependent manner, as well as migrative activity of EPCs in vitro (p<0.05). The co-treatment of Phentolamine, I127, LY294002 and L-NAME with NE blocked the effects of NE on EPCs proliferation and migration. Treatment with NE significantly increased phosphorylation of Akt and eNOS of EPCs. Addition of phentolamine and I127 attenuated the activation of Akt/eNOS pathway, but metoprolol could not. Pretreatment of mice with either Phentolamine or I127 significantly attenuated the effects of NE on EPCs in vivo, VEGF concentration in BM, skeletal muscle and angiogenesis in ischemic gastrocnemius, but Metoprolol did not.ConclusionThese results unravel that sympathetic nervous system regulate EPCs mobilization and their pro-angiogenic capacity via α adrenoceptor, β 2 adrenoceptor and meanwhile Akt/eNOS signaling pathway.

Possible pathogenic role of T helper type 9 cells and interleukin (IL)-9 in atopic dermatitis

T helper type 9 (Th9) cells are a novel identified subset of CD4(+) T helper cells, which could partly contribute to allergic inflammation, while the precise contribution of Th9 cells in atopic dermatitis (AD) remains unknown. We aimed to explore the possible role of Th9 cells in AD pathogenesis. The Th9 cell percentage, transcription factor PU.1 and cytokine interleukin (IL)-9 mRNA levels, as well as IL-9 serum concentration in peripheral circulation, were measured in AD patients, psoriasis patients and healthy controls. The Th9 cell percentage, PU.1 and IL-9 expression levels of AD patients were all increased significantly compared with the other two control groups (P < 0·01), and correlated positively with SCORing Atopic Dermatitis index, serum immunoglobulin (Ig)E and thymus- and activation-regulated chemokine (TARC) levels (P < 0·05). In simple AD patients and AD patients complicated by allergic rhinitis or asthma, there were no significant differences in the Th9 cell percentage, PU.1 and IL-9 expression levels between them. At the same time, IL-9 and vascular endothelial growth factor (VEGF) mRNA levels were detected in AD lesions and normal skin samples, which were both distinctly elevated in AD lesions, and there was a positive association between them (P < 0·01). Keratinocytes were cultured with IL-9 stimulation and the secretion of VEGF was detected. IL-9 can promote the secretion of VEGF by keratinocytes in a time- and dose-dependent manner. In conclusion, the expansion of the Th9 cell subset, up-regulation of the PU.1 transcription factor and increased secretion of the IL-9 cytokine may contribute to the pathogenesis of AD, which may be supported by the increased release of VEGF by keratinocyes after IL-9 stimulation.

Detection and identification of plasma progesterone metabolites in the female Florida manatee ( Trichechus manatus latirostris) using GC/MS/MS

Florida manatees ( Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants—terrestrial manatee relatives—were targeted. These included 5α-reduced progestins (5α-pregnane-3,20-dione [5α-DHP] and 3α-hydroxy-5α-pregnan-20-one [5α-P3-OH]) and 17α-hydroxyprogesterone (17α-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20α-hydroxyprogesterone (20α-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography–mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5α-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5α-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17α-OHP metabolite was not detected in any tested female. The 20α-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.

Altered cytokine levels in the blood and cerebrospinal fluid of chronic pain patients

This study replicates and extends prior reports of abnormal cytokine levels in chronic pain patients and has correlated the alterations with pain severity. In addition, there appeared to be a need to directly assess cerebrospinal fluid (CSF) because previous findings on cytokine concentrations in peripheral circulation have been inconsistent. CSF and blood specimens were obtained from 14 patients with distal painful non-diabetic polyneuropathy (DPPN) or post-traumatic neuralgia (PTN). Elevated receptor levels for Tumor Necrosis Factor (sTNFr) were the most distinctive abnormality along with low interleukin-10 (IL-10). sTNFr in CSF and blood, and IL-1ß in CSF, were positively associated with pain intensity, whereas IL-10 in both compartments was inversely correlated with pain symptoms. An imbalance of pro- and anti-inflammatory cytokines appears to be a clinically relevant feature, which may contribute to the maintenance of chronic pain.

Non-invasive methods of assessing angiogenesis and their value in predicting response to treatment in colorectal cancer.

Tumour neoangiogenesis can be assessed non-invasively by measuring angiogenic cytokine concentrations in peripheral circulation and by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). The aim of this study was to assess whether these methods can predict and monitor response to treatment in patients with rectal cancer treated with preoperative chemoradiotherapy. Serum and plasma vascular endothelial growth factor levels were measured in 31 patients with T3/T4 rectal cancers before quantitating tumour permeability (ln Ktrans) by DCE-MRI. Sixteen patients receiving preoperative chemoradiotherapy had serial vascular endothelial growth factor (VEGF) and DCE-MRI measurements. Response to treatment was assessed using World Health Organization criteria. Serum VEGF and ln Ktrans correlated before treatment (r = 0.48, P = 0.01). Responsive tumours (n = 8) had higher pretreatment permeability values than non-responsive tumours (n = 8) (mean ln Ktrans - 0.46 and - 0.72 respectively; P = 0.03). Compared with pretreatment values, responsive tumours showed a marked reduction in permeability at the end of treatment (mean ln Ktrans - 0.46 and - 0.86 respectively; P = 0.04). Pretreatment serum VEGF levels were not statistically different between the two groups. Rectal tumours with higher permeability at presentation appear to respond better to chemoradiotherapy than those of lower permeability. This may allow preselection of appropriate tumours for these regimens, with patients with low-permeability tumours being considered for alternative therapies.

Growth hormone secretion in the horse: unusual pattern at birth and pulsatile secretion through to maturity.

A heterologous radioimmunoassay was developed and validated for the measurement of horse GH in plasma. It utilized recombinant-derived bovine GH as the radiolabelled ligand, a guinea-pig anti-porcine GH serum as first antibody and pituitary-derived horse GH as standard. Cross-reactivities were high with all of the pituitary and recombinant-derived GH preparations tested (49-140%) and very low (< 0.3%) with horse FSH, LH and prolactin. A synthetic analogue of GH-releasing factor(1-29) stimulated the expected pattern of GH release in foals. Plasma GH concentrations in foals were low at birth (< 20 ng/ml) but rose sharply to a definite and, in most cases, very large peak (18-195 ng/ml) during the first 30-40 min post partum, followed by a steady decline to basal levels again by 60-100 min post partum. GH secretion was clearly pulsatile in all older foals tested (2 weeks, 1 month and 4 months of age) and in six adults (three mares and three stallions), all bled at 15-min intervals for 7-8 h. Basal levels and pulse amplitudes were higher in foals than in adults and pulse frequency was higher in stallions than in mares (3-5 pulses/8 h vs 1-2 pulses/8 h). Pulsatile secretion was further characterized in one mare by simultaneous sampling of jugular vein and pituitary cavernous sinus blood. Peak GH concentrations in cavernous sinus blood draining the pituitary gland were more than tenfold higher than the corresponding peak concentrations in peripheral circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of age and intake on growth hormone kinetics in dairy heifers

The effects of aging and intake on growth hormone (GH) kinetics and GH-releasing factor (GRF)-induced GH concentrations were studied in two groups of 12 Holstein heifers each (80 d, 85 kg: young; and 273 d of age, 246 kg: old). Each group was then equally subdivided into full-fed (FF) and restricted-fed (RF) subgroups. After 11 d of intake treatment, animals were infused for 3 hr with GH (1.5 mg/hr) in order to calculate GH metabolic clearance rate (MCR), secretion rate (SR) and half-life ( t 1 2 ). Two d later, total plasma volume was determined and the following day, all heifers received a GRF challenge (5 μg/kg i.v.). The following values are LSM ± SE for young-FF, young-RF, old-FF and old-RF. Rate of secretion was not affected by any treatment, averaging 1.51, 1.25, 1.34, and 1.40 ± .23 μg/min. Aging increased (P<.01) MCR (186, 159, 382, and 300 ± 21 ml/min) and increased plasma volume (P<.01), which resulted in lower basal GH concentrations. Aging also decreased (P<.01) the area under the GH response curve following GRF injection (AUC: 12442, 21114, 5155, and 6308 ± 1776 ng · min/ml) but did not affect average GH quantity in the plasma after the GRF challenge. Feed restriction decreased (P<.05) MCR, but not enough to affect basal GH concentrations. However, feed restriction increased GRF-induced GH concentrations in young heifers only; since all GH kinetic variables were similarly affected by intake in old and young heifers, this effect of feed restriction in the young heifers probably resulted from a higher pituitary secretion of GH when challenged with GRF. Concentrations of insulin, glucagon, glucose, insulin-like growth factor-I (IGF-I) and free fatty acids (FFA) were determined. There was a correlation (P<.001) between IGF-I and GH MCR (r 2=.65) and AUC (r 2=−.66), and a correlation between FFA and GH secretion rate (r 2=−.41, P<.05). The AUC was related to the MCR (r 2=−.67, P<.001). These data therefore indicate that post-secretory metabolism of GH plays an important role in determining GH concentrations in peripheral circulation. The effect of aging (from 3 to 9 mo) on GH concentrations is mostly due to increased MCR and plasma volume, while feed restriction affects the kinetics of GH and the GRF-induced secretion.

Inhibition of Pituitary Gonadotropin Secretion by the Gonadotropin-Releasing Hormone Antagonist Antide. II. Development of an in Vitro Bioassay for Characterization of Pharmacokinetics and Pharmacodynamics of Antide in Circulation*

Previous data from this laboratory revealed a rapid and unexpectedly long inhibition of pituitary gonadotropin secretion in ovariectomized monkeys after a single high dose injection of the GnRH antagonist antide. This extended action of antide may correlate with an extended presence of antide in the peripheral circulation. We have reported on use of a RRA for antide in serum; however, during such a prolonged presence in the body, the possibility of catabolic loss of biological activity remained to be analyzed. In the present study, we have developed an in vitro pituitary cell bioassay for antide to investigate the pharmacokinetics and possible mechanism(s) contributory to its long action. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and incubated with 0.024-6 ng antide for 24 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide standards or serum samples for 4 h. Before antide injection into long term ovariectomized monkeys, peripheral GnRH antagonist levels were undetectable. One day after a single injection (3.0 mg/kg, sc, in 50% propylene glycol-water), the level of antide was 31 +/- 13 ng/ml (n = 3). Thereafter, antide levels declined slowly and were still detectable (greater than 1.4 ng/ml) in two of three monkeys 31 days after injection. After iv administration (3.0 mg/kg; n = 2), peripheral antide levels followed a similar pharmacokinetic profile and declined slowly. Detectable antide concentrations were still present 36 days after single iv injection in both monkeys. The circulating half-lives of antide were 1.7 and 14.5 days for the first and second phases, respectively. Peripheral LH levels were suppressed to the limits of detectability within 1 day and slowly recovered to pretreatment levels within 30 +/- 5 days after sc or iv antide treatment. The ratio of bioactive antide to antide levels measured by RRA was similar throughout the study (chi = 1.24 +/- 0.09; range, 0.40-2.22), although there was a trend toward an increased B/R ratio at the end of the study. In summary, we have developed an in vitro bioassay using cultured rat pituitary cells to measure biologically active antide concentrations in peripheral circulation after sc and iv treatments. The prolonged action of antide on pituitary gonadotropin secretion in vivo is apparently due to the continued presence of biologically active antide in circulation after a single injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Active immunization of gilts against gonadotropin-releasing hormone: effects on secretion of gonadotropins, reproductive function, and responses to agonists of gonadotropin-releasing hormone.

Sexually mature gilts were actively immunized against gonadotropin-releasing hormone (GnRH) by conjugating GnRH to bovine serum albumin, emulsifying the conjugate in Freund's adjuvant, and giving the emulsion as a primary immunization at Week 0 and as booster immunizations at Weeks 10 and 14. Antibody titers were evident by 2 wk after primary immunization and increased markedly in response to booster immunizations. Active immunization against GnRH caused gonadotropins to decline to nondetectable levels, gonadal steroids to decline to basal levels, and the gilts to become acyclic. Prolactin concentrations in peripheral circulation were unaffected by immunization against GnRH. The endocrine status of the hypothalamic-pituitary-ovarian axis was examined by giving GnRH and two agonists to GnRH and by ovariectomy. An i.v. injection of 100 micrograms GnRH caused release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in control animals, but not in gilts immunized against GnRH. In contrast, administration of 5 micrograms D-(Ala6, des-Gly-NH2(10] ethylamide or 5 micrograms D-(Ser-t-But6, des-Gly-NH2(10] ethylamide resulted in immediate release of LH and FSH in both control and GnRH-immunized gilts. Circulating concentrations of LH and FSH increased after ovariectomy in the controls, but remained at nondetectable levels in gilts immunized against GnRH. Prolactin concentrations did not change in response to ovariectomy. We conclude that cyclic gilts can be actively immunized against GnRH and that this causes cessation of estrous cycles and inhibits secretion of LH, FSH, and gonadal steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of the onset of puberty in the female rat

The effect of 5α-androstane-3α, 17β-diol (3α-androstanediol) and its 3β epimer on the mechanism of the onset of puberty has been investigated. In the course of the study it was found that in two strains of rats examined, in the majority of animals vaginal opening is not accompanied by ovulation, nor is the presence of corpora lutea an indication for the first ovulation, whereas significant uterine weight increase is a correlate for the first presence of tubal ova. The first preovulatory LH surge was also found to occur on the day preceding the first cornified smear which was not the day of vaginal opening.A daily dose of 25 μg of 3⊝androstanediol from the 21st day up to vaginal opening advanced the first ovulation. 3α-androstanediol was found to be effective in inhibiting postcastrational LH release in immature female rats, while the 3β epimer was inactive in this respect. The 3β epimer advanced vaginal opening by an effect mediated by the ovaries. The origin of the secretion of these steroids was found to be ovarian, since ovariectomy reduced their concentration in peripheral circulation to an undetectable level. The possibility that these compounds participate in the mechanism of the onset of puberty is discussed.