ABSTRACT

The acute effects of different macronutrients on the secretion of glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) and glucose-dependent insulinotropic polypeptide (GIP) were compared in healthy human subjects. Circulating levels of the two hormones were measured over a 24-h period during which subjects consumed a mixed diet. In the first study, eight subjects consumed three equicaloric (375 kcal) test meals of carbohydrate, fat and protein. Small increases in plasma GLP-1(7–36) amide were found after all meals. Levels reached a maximum 30 min after the carbohydrate and 150 min after the fat load. Ingestion of both carbohydrate and fat induced substantial rises in GIP secretion, but the protein meal had no effect. In a second study, eight subjects consumed 75 g glucose or the equivalent portion of complex carbohydrate as boiled brown rice or barley. Plasma GIP, insulin and glucose levels increased after all three meals, the largest increase being observed following glucose and the smallest following the barley meal. Plasma GLP-1(7–36)amide levels rose only following the glucose meal. In the 24-h study, plasma GLP-1(7–36)amide and GIP concentrations were increased following every meal and remained elevated throughout the day, only falling to fasting levels at night. The increases in circulating GLP-1(7–36)amide and GIP levels following carbohydrate or a mixed meal are consistent with their role as incretins. The more sustained rises observed in the daytime during the 24-h study are consistent with an anabolic role in lipid metabolism.

Journal of Endocrinology (1993) 138, 159–166

SALL1 was originally identified on the basis of its DNA sequence homology to the region-specific homeotic gene Sal, in Drosophila melanogaster, which acts as a downstream target of hedgehog/tumor growth factor-beta-like decapentaplegic signals. The SALL1 gene has been associated with the Townes-Brocks Syndrome (TBS), a disorder characterized by multiorgan dysgenesis including renal and genital malformations. In this study, SALL1 message production was evaluated in association with the tissue localization of the protein product of SALL1, p140. SALL1 protein expression was observed in various adult and fetal tissues which elaborate reproductive endocrine hormones. The p140 was localized in specific microanatomic sites of the pituitary, adrenal cortex and the placenta. In the human pituitary, SALL1 protein expression was limited to the adenohypophysis, where it colocalized to those cells producing GH and the gonadotropins, LH and FSH. SALL1 expression was also found in most of the fetal and adult adrenal cortex in addition to the trophoblastic cells of the placenta. This pattern of expression complements prior studies demonstrating p140 in testicular fetal Leydig cells, adult Leydig and Sertoli cells, and granulosa cells of the ovary. The SALL1 protein was also shown here to be highly expressed in trophoblast tumors, which overproduce sex hormones. The expression patterns of SALL1 at multiple levels of the reproductive endocrine axis and the phenotypic effects associated with TBS suggest that SALL1 may have an important role in the interaction of the pituitary-adrenal/gonadal axis during reproduction.

ABSTRACT

The induction of ovulation in early post-partum ewes is associated with a high incidence of premature luteal regression which is independent of the suckling stimulus but dependent on the stage post partum. The aim of the present study was to determine whether oxytocin receptors are present on uterine endometrium early in the luteal phase and hence ascertain whether oxytocin-induced uterine prostaglandin F_2α release is a possible mechanism involved in the premature regression of these post-partum corpora lutea. Ovarian and uterine tissues were collected on day 4 of the cycle in ewes induced to ovulate at either 21 or 35 days post partum (n = 4 per group). A further four cyclic ewes were similarly synchronized to ovulate and acted as controls. Corpora lutea from the 21-day post-partum group were significantly (P < 0·01) smaller, had a lower progesterone content and a reduced capacity to secrete progesterone in vitro than corpora lutea from 35-day post-partum or control ewes.

A highly specific oxytocin receptor ligand ¹²⁵I-labelled d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH₂ ⁹]-vasotocin was used to localize and characterize high affinity oxytocin receptors in uterine endometrium (dissociation constant 145 pmol/l). Oxytocin receptor concentrations in endometrium from ewes induced to ovulate at 21 days post partum were on average five-fold higher (P < 0·05) than in 35-day post-partum and control groups.

Journal of Endocrinology (1991) 128, 253–260

ABSTRACT

The effects of the mixed α/β-agonist adrenaline on insulin secretion from isolated human islets of Langerhans were studied. In static incubation experiments, adrenaline (0·1 nmol/l to 10 μmol/l) caused a concentration-dependent inhibition of glucose-induced insulin secretion from isolated human islets. However, perifusion experiments revealed that the time-course of the secretory changes induced by adrenaline was complex. When employed at a high concentration (1 μmol/l), adrenaline caused a sustained inhibition of glucose-induced insulin secretion, which could be relieved by the addition of the α₂-antagonist yohimbine (10 μmol/l). By contrast, infusion of adrenaline at a lower concentration (10 nmol/l), produced a large initial potentiation of glucose-induced insulin secretion. This response was, however, short-lived and followed by sustained inhibition of secretion, which could be relieved by yohimbine (10 μmol/l). The initial stimulation of insulin secretion provoked by 10 nmol adrenaline/l was abolished when islets were incubated in the presence of the β-antagonist, propranolol (1 μmol/l), consistent with activation of β-adrenoceptors. In support of this, treatment of human islets with the selective β₂-agonist clenbuterol, was also associated with marked stimulation of insulin secretion. By contrast, each of two selective β₃-agonists tested failed to alter insulin secretion from human islets. The results indicate that human pancreatic B-cells are equipped with both α₂-and β₂-adrenoceptors which can affect insulin secretion. Adrenaline interacts with both of these but the α₂-response is predominant and can overcome the tendency of β₂-adrenoceptors to potentiate insulin release.

Journal of Endocrinology (1993) 138, 555–563

The aged phenotype shares several metabolic similarities with that of circulatory glucocorticoid excess (Cushing’s syndrome), including type 2 diabetes, obesity, hypertension, and myopathy. We hypothesise that local tissue generation of glucocorticoids by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts 11-dehydrocorticosterone to active corticosterone in rodents (corticosterone to cortisol in man), plays a role in driving age-related chronic disease. In this study, we have examined the impact of ageing on glucocorticoid metabolism, insulin tolerance, adiposity, muscle strength, and blood pressure in both wildtype (WT) and transgenic male mice with a global deletion of 11β-HSD1 (11β-HSD1^−/−) following 4 months high-fat feeding. We found that high fat-fed 11β-HSD1^−/− mice were protected from age-related glucose intolerance and hyperinsulinemia when compared to age/diet-matched WTs. By contrast, aged 11β-HSD1^−/− mice were not protected from the onset of sarcopenia observed in the aged WTs. Young 11β-HSD1^−/− mice were partially protected from diet-induced obesity; however, this partial protection was lost with age. Despite greater overall obesity, the aged 11β-HSD1^−/− animals stored fat in more metabolically safer adipose depots as compared to the aged WTs. Serum analysis revealed both WT and 11β-HSD1^−/− mice had an age-related increase in morning corticosterone. Surprisingly, 11β-HSD1 oxo-reductase activity in the liver and skeletal muscle was unchanged with age in WT mice and decreased in gonadal adipose tissue. These data suggest that deletion of 11β-HSD1 in high fat-fed, but not chow-fed, male mice protects from age-related insulin resistance and supports a metabolically favourable fat distribution.

The adverse metabolic effects of prescribed and endogenous glucocorticoid excess, ‘Cushing’s syndrome’, create a significant health burden. While skeletal muscle atrophy and resultant myopathy is a clinical feature, the molecular mechanisms underpinning these changes are not fully defined. We have characterized the impact of glucocorticoids upon key metabolic pathways and processes regulating muscle size and mass including: protein synthesis, protein degradation, and myoblast proliferation in both murine C2C12 and human primary myotube cultures. Furthermore, we have investigated the role of pre-receptor modulation of glucocorticoid availability by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in these processes. Corticosterone (CORT) decreased myotube area, decreased protein synthesis, and increased protein degradation in murine myotubes. This was supported by decreased mRNA expression of insulin-like growth factor (IGF1), decreased activating phosphorylation of mammalian target of rapamycin (mTOR), decreased phosphorylation of 4E binding protein 1 (4E-BP1), and increased mRNA expression of key atrophy markers including: atrogin-1, forkhead box O3a (FOXO3a), myostatin (MSTN), and muscle-ring finger protein-1 (MuRF1). These findings were endorsed in human primary myotubes, where cortisol also decreased protein synthesis and increased protein degradation. The effects of 11-dehydrocorticosterone (11DHC) (in murine myotubes) and cortisone (in human myotubes) on protein metabolism were indistinguishable from that of CORT/cortisol treatments. Selective 11β-HSD1 inhibition blocked the decrease in protein synthesis, increase in protein degradation, and reduction in myotube area induced by 11DHC/cortisone. Furthermore, CORT/cortisol, but not 11DHC/cortisone, decreased murine and human myoblast proliferative capacity. Glucocorticoids are potent regulators of skeletal muscle protein homeostasis and myoblast proliferation. Our data underscores the potential use of selective 11β-HSD1 inhibitors to ameliorate muscle-wasting effects associated with glucocorticoid excess.

Abstract

This study was designed to investigate postprandial responses to a mixed meal in simulated shift work conditions. Nine normal healthy subjects (six males and three females) were studied on two occasions at the same clock time (1330 h) after consuming test meals, first in their normal environment and secondly after a 9 h phase advance (body clock time 2230 h). Plasma glucose, insulin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), triacylglycerol (TAG) and non-esterified fatty acids (NEFAs) were determined at intervals for 6 h after each test meal. Postprandial plasma glucose, insulin, GIP and GLP-1 profiles were evaluated by calculating areas under the curve (AUC) for the first 2 h and the last 4 h of the sampling together with total AUC. Significantly higher postprandial glucose responses (total AUC) were observed after the phase shift than before (AUC 0–360 min, 2·01 (1·51–2·19) vs 1·79 (1·56–2·04) mmol/l.min; P<0·02; mean (range)). No significant difference was observed when the first 2 h of each response was compared, but significantly higher glucose levels were observed in the last 4 h of the study after the phase shift than before (AUC 120–360 min, 1·32 (1·08–1·42) vs 1·16 (1·00–1·28) mmol/l.min; P<0·05). Similar results were obtained for insulin (AUC 0—360 min, 81·72 (30·75– 124·97) vs 58·98 (28·03–92·57) pmol/l.min; P<0·01; AUC 120–360 min, 40·73 (16·20–65·25) vs 25·71 (14·25–37·33) pmol/l.min; P<0·02). No differences were observed in postprandial plasma GIP and GLP-1 responses before and after the phase shift. Postprandial circulating lipid levels were affected by phase shifting. Peak plasma TAG levels occurred 5 h postprandially before the phase shift. Postprandial rises in plasma TAG were significantly delayed after the phase shift and TAG levels continued to rise throughout the study. Plasma postprandial NEFA levels fell during the first 3 h both before and after the phase shift. Their rate of return to basal levels was significantly delayed after the phase shift compared with before. This study demonstrates that a simulated phase shift can significantly alter pancreatic B-cell responses and postprandial glucose and lipid metabolism.

Journal of Endocrinology (1996) 151, 259–267

This study was undertaken to determine whether the internal clock contributes to the hormone and metabolic responses following food, in an experiment designed to dissociate internal clock effects from other factors. Nine female subjects participated. They lived indoors for 31 days with normal time cues, including the natural light: darkness cycle. For 7 days they retired to bed from 0000 h to 0800 h. They then underwent a 26-h 'constant routine' (CR) starting at 0800 h, being seated awake in dim light with hourly 88 Kcal drinks. They then lived on an imposed 27-h day (18 h of wakefulness, 9 h allowed for sleep), for a total of 27 days. A second 26-h CR, starting at 2200 h, was completed. During each CR salivary melatonin and plasma glucose, triacylglycerol (TAG), non-essential fatty acids (NEFA), insulin, gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) were measured hourly. Melatonin and body temperature data indicated no shift in the endogenous clock during the 27-h imposed schedule. Postprandial NEFA, GIP and GLP-1 showed no consistent effects. Glucose, TAG and insulin increased during the night in the first CR. There was a significant effect of both the endogenous clock and sleep for glucose and TAG, but not for insulin. These findings may be relevant to the known increased risk of cardiovascular disease amongst shift workers.

Abstract

Sequences from cDNA molecules encoding α₂-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human α₂C2-, α₂C4- and α₂C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either ³²P or digoxigenin and used to study the expression of α₂-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic β-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both α₂C2 and α₂C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding α₂C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each α₂-adrenoceptor subtype in sections of human pancreas. All three subtypes of α₂-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffinembedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three α₂-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple α₂-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of α₂-adrenoceptor subtype mRNA species in pancreatic β-cells was confirmed by Northern blotting of RNA extracted from the clonal β-cell line, HIT-T15. Transcripts encoding each of the three cloned α₂-adrenoceptor subtypes were detected in HIT-T15 cells.

Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with β₂-adrenoceptor mRNA revealed expression of this species in islet β-cells but not in the exocrine tissue of the pancreas.

Journal of Endocrinology (1996) 148, 531–543

ABSTRACT

Two forms of inhibin with molecular weights of 65 000 and 30 000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in >95% purity (1210-fold purification and 4·2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20–21 and 16 kD of which the 20–21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin.

J. Endocr. (1987) 113, 213–221