The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor 1a (GHS-R1a), and consists of 28 amino acid residues with octanoyl modification at Ser³. The previous studies have revealed that N-terminal part of ghrelin including modified Ser³ is the active core for the activation of GHS-R1a. On the other hand, the role of C-terminal (8-28) region in ghrelin has not been clarified yet. In the present study, we prepared human ghrelin, C-terminal truncated ghrelin derivatives and anamorelin, a small molecular GHS compound which supposedly mimics the N-terminal active core, and examined GHS-R1a agonist activity in vitro, pharmacokinetic (PK) profile and growth hormone (GH) releasing activity in rats. All compounds demonstrated potent GHS-R1a agonist activities in vitro. Although the lack of C-terminal two amino acids did not modify PK profile and GH releasing activity, the deletion of C-terminal 8 and 20 amino acids affected them, and ghrelin(1-7)-Lys-NH₂ exhibited very short plasma half-life and low GH releasing activity in vivo. In rat plasma, ghrelin(1-7)-Lys-NH₂ was degraded more rapidly than ghrelin, suggesting that C-terminal part of ghrelin protected octanoylation of Ser³ from plasma esterases. Subdiaphragmatic vagotomy significantly attenuated GH response to ghrelin but not to anamorelin. These results suggest that the C-terminal part of ghrelin has an important role in the biological activity in vivo. We also found that ghrelin stimulated GH release mainly via a vagal nerve pathway but anamorelin augmented GH release possibly by directly acting on brain in rats.     

Urinary free cortisone, but not cortisol, is associated with urine volume in severe obesity

Background

High urine volume enhances urinary free cortisol (UFF) and cortisone (UFE) excretion rates in normal-weight adults and children. Renal excretion rates of glucocorticoids (GC) and their metabolites are frequently altered in obesity. The aim of the present study was to investigate whether UFF and UFE excretion is also affected by urine volume in severely obese subjects.

Experimental

In 24-h urine samples of 59 extremely obese subjects (mean BMI 45.3 ± 8.9 kg/m²) and 20 healthy lean subjects (BMI 22.1 ± 1.8 kg/m²), UFF and UFE, tetrahydrocortisol (THF), 5α-tetrahydrocortisol (5α-THF), and tetrahydrocortisone (THE) were quantified by RIA. The sum of THF, 5α-THF, and THE (GC3), the three major GC metabolites, reflects daily cortisol secretion. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity was assessed by the ratio UFE/UFF. Daily GC excretion rates were corrected for urine creatinine and adjusted for gender and body weight.

Results

In extremely obese subjects, urine volume was significantly associated with creatinine-corrected UFE and 11β-HSD2 activity after adjustment for gender and BMI (r = 0.47, p = 0.0002 and r = 0.31, p = 0.02, respectively). However, urine volume was not associated with creatinine-corrected UFF and GC3 (p = 0.4 and p = 0.6, respectively). In lean controls, urine volume was significantly associated with creatinine-corrected UFE and UFF (r = 0.58, p = 0.01 and r = 0.55, p = 0.02, respectively), whereas urine volume was not associated with 11β-HSD2 activity after appropriate adjustment (p = 0.3).

Conclusions

In severe obesity, in contrast to normal weight, renal excretion of UFE, but not of UFF is affected by fluid intake. This discrepancy may be due to the increased renal 11β-HSD2 activity in obesity.     

Non-invasive monitoring of the estrous cycle in captive crab-eating foxes (Cerdocyon thous)

In this study, four crab-eating fox females (Cerdocyon thous) maintained at the Federal University of Mato Grosso Zoo, Cuiabá, Brazil, were investigated for 16 mo, using transabdominal ultrasonography and measurement of estradiol and progesterone concentrations in blood plasma and feces. Blood collection and ultrasonography were performed once a month, whereas fecal collections were performed three times a week. During the experimental period, there was an annual estrous cycle in all females, with the reproductive season lasting from winter to spring, and three became pregnant. Transabdominal ultrasonography was inconclusive for characterization of estrus cycles phase, but was effective for early detection of pregnancy, pregnancy monitoring, and for evaluating postpartum uterine involution. There were similarities between C. thous female's reproductive aspect and bitches, with similar pregnancy data, although uterine involution was faster in C. thous. Peak serum concentrations of P4 and E2 were (mean ± SD) 14.58 ± 5.8 ng/ml and 31.62 ± 53.54 pg/ml, respectively, whereas mean fecal peaks of P4 and E2 were 2.37 ± 1.42 ng/g and 157.95 ± 82.63 pg/g, respectively. All pregnant females had serum and fecal P4 concentrations reaching maximum values (16.5 ± 4.0 ng/ml and 2.7 ± 0.4 ng/g, respectively) from 10 to 30 d of gestation; those values subsequently declined, reaching baseline at parturition (5.0 ± 4.0 and 0.7 ± 0.4 ng/g, respectively). Peaks of E2 occurred throughout the year, and were absent only during apparent lactational anestrus.     

Effects of noradrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intra-adrenal paracrine interactions

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo noradrenaline (NA) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, NA, and adrenaline (A). In March and July, NA administration increased aldosterone release (from 187.23 +/- 2.93 pg/ml to 878.31 +/- 6.13 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 622.51 +/- 2.65 pg/ml in July) from steroidogenic cells. The cells showed clear signs of stimulation, as evidenced by a strong reduction of lipid content. Moreover, NA administration decreased the mean total number of secretory vesicles in the chromaffin cells in March (from 7.24 +/- 0.18 granules/micro2 to 5.57 +/- 1.88 granules/micro2) and July (from 7.74 +/- 0.74 granules/micro2 to 6.04 +/- 1.13 granules/micro2). In March, however, when T. carnifex chromaffin cells contain both catecholamines, NA (3.88 +/- 0.13 granules/micro2) and A (3.36 +/- 0.05 granules/micro2) in almost equal quantities, NA administration reduced A content (1.29 +/- 1.04 granules/micro2) in the chromaffin cells, enhancing adrenaline secretion (from 681.27 +/- 1.83 pg/ml to 1527.02 +/- 2.11 pg/ml). In July, when the chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/micro2; A: 0.32 +/- 0.13 granules/micro2), NA administration reduced the number of NA granules (5.45 +/- 1.10 granules/micro2), thereby increasing noradrenaline release from the chromaffin cells (from 640.19 +/- 1.65 pg/ml to 1217.0 +/- 1.14 pg/ml). The results of this study indicate that NA influences the steroidogenic cells, eliciting aldosterone release. Noradrenalin effects on the chromaffin cells, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the steroidogenic cells. The existence of intra-adrenal paracrine interactions in T. carnifex is discussed.     

Effects of adrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intraadrenal paracrine interactions

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo adrenaline (A) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, noradrenaline (NA), and adrenaline. In March and July, adrenaline administration reduced aldosterone release (from 187.23 +/- 2.93 pg/ml to 32.28 +/- 1.85 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 87.51 +/- 2.57 pg/ml in July) from steroidogenic cells. The cells showed clear signs of lowered activity: they appeared full of lipid, forming large droplets. Moreover, adrenaline administration decreased the mean total number of secretory granules in the chromaffin cells in July (from 7.74 +/- 0.74 granules/microm(2) to 5.14 +/- 1.55 granules/microm(2)). In this period T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm(2); A: 0.32 +/- 0.13 granules/microm(2)). Adrenaline administration reduced noradrenaline content (4.36 +/- 1.40 granules/microm(2)) in the chromaffin cells, enhancing noradrenaline secretion (from 640.19 +/- 1.65 pg/ml to 1030.16 +/- 3.03 pg/ml). In March, adrenaline administration did not affect the mean total number of secretory vesicles (from 7.24 +/- 0.18 granules/microm(2) to 7.25 +/- 1.97 granules/microm(2)). In this period the chromaffin cells contain both catecholamines, noradrenaline (3.88 +/- 0.13 granules/microm(2)), and adrenaline (3.36 +/- 0.05 granules/microm(2)), in almost equal quantities; adrenaline administration reduced adrenaline content (1.74 +/- 0.84 granules/microm(2)), increasing adrenaline release (from 681.27 +/- 1.83 pg/ml to 951.77 +/- 4.11 pg/ml). The results of this study indicate that adrenaline influences the steroidogenic cells, inhibiting aldosterone release. Adrenaline effects on the chromaffin cells (increase of noradrenaline or adrenaline secretion) vary according to the period of chromaffin cell functional cycle. The existence of intraadrenal paracrine interactions in T. carnifex is discussed.     

Synthetic insulin-like growth factor II

Human insulin-like growth factor II with 67 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. Homogeneity of the synthetic product is ascertained by chromatofocusing, high performance liquid chromatography and amino acid analysis. In both radioimmunoassay and radioreceptor assay, the synthetic product is indistinguishable from the natural hormone.     

Monitoring of individual human exposure to aflatoxins (AF) and N-nitrosamines (NNO) by immunoassays

Highly sensitive immunoassays have been used to quantitate aflatoxins (AF) and N-nitrosamines (NNO) in human body fluids and tissues, respectively. This approach was taken in order to quantitate environmental exposure to these agents at an individual level to facilitate the investigation of their role in the etiology of human cancer. In order to analyse AF in human urine, an immunopurification step has been developed by using AF-specific antibody bound to AH-Sepharose 4B gel in a small (4-ml gel volume) affinity column prior to enzyme-linked immunosorbent assay (ELISA). The ELISA can be used to quantitate aflatoxin B1 (AFB1) over the range 0.01 ng/ml to 10 ng/ml and the assay system has been validated by using human urine samples spiked with AFB1 over this concentration range. In addition, 29 urine samples from the Philippines have been analysed and found to contain a range of levels from zero to 4.25 ng/ml AFB1 equivalent with a mean of 0.875 ng/ml. This compared with a mean of 0.066 ng/ml AFB1 equivalent in samples from France. Radioimmunoassay of O6-methyldeoxyguanosine (O6-medG) has been performed on human oesophageal and cardiac stomach mucosal DNA from tissue samples obtained during surgery in Linxian County, People's Republic of China, an area of high risk for both oesophageal and stomach cancer. Using the methodology described and having 1 mg of hydrolyzed DNA allows the detection of approximately 25 fmol O6medG per mg DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha-melanocyte-stimulating hormone in rat pancreas

Reverse-phase high performance liquid chromatography and radioimmunoassay were used to characterize alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in rat pancreas. Relative to synthetic alpha-MSH standards, serial dilutions of pancreas extracts showed parallel and concentration dependent displacement of (125I) alpha-MSH from alpha-MSH antibody. Chromatographic separation revealed immunoreactive material coeluting with synthetic N,O-diacetyl alpha-MSH, which accounted for 78% of total alpha-MSH materials in this tissue. The remainder of immunoreactive alpha-MSH coeluted with synthetic alpha-MSH, desacetyl alpha-MSH, or their methionine sulfoxides. In contrast with anterior pituitary, it appears that biosynthetic processing of alpha-MSH from pro-opiomelanocortin (POMC) may be similar in rat pancreas and pituitary intermediate lobe, since their relative alpha-MSH immunoreactive elution profiles were similar. These findings support the hypothesis of tissue specific regulation of biosynthetic processing of POMC.     

动脉壁脑啡肽的含量,存在部位及作用途径

用放射免疫法测得兔动脉的亮氨酸脑啡肽(L-ENK)和甲硫氨酸脑啡肽(M-ENK)样免疫活性物的含量(pg/mg组织湿重)分别为耳动脉38.99±17.29,134.67±8.11;肾动脉31.10±7.76,93.60±18.22,肠系膜动脉25.70 13.60,88.43±18.16。动物经注射交感神经末梢化学切割剂6-OHDA后,这三种动脉中L-ENK样免疫活性物的含量均明显下降(P<0.001);切除颈上神经节使支配耳动脉的交感神经溃变后,或离体耳动脉条受电场刺激后,脑啡肽(ENK)样免疫活性物的含量也都显著下降(P<0.001);但注射利血平则无明显影响,用荧光法测定培育动脉条的浴槽液中NE的含量,观察到ENK可抑制电场刺激所引起的NE的释放。结果提示,动脉壁的L-ENK和M-ENK存在于交感神经末梢中,它可因受电场刺激而释放,可能通过激活突触前膜阿片受体,减少交感神经末梢释放NE,从而抑制动脉的收缩活动。