ABSTRACT

Suckling rats exhibit age-dependent differences in epidermal growth factor (EGF) levels in several organs. The present studies evaluated the effects of two hormones known for their maturative effect on suckling rats, cortisone and tri-iodothyronine (T₃), on immunoreactive EGF levels in specific organs. Suckling rats were administered cortisone (5 mg/100 g body weight per day) or T₃ (50 μg/100 g body weight per day) on days 8, 9, 10 and 11 after birth, and killed on day 12. Submandibular glands, kidneys, pancreas, liver and gastrointestinal tract mucosa and lumen were assayed for immunoreactive EGF by a speciesspecific radioimmunoassay. Low levels of EGF in the submandibular glands were increased slightly by both T₃ and cortisone treatment. Cortisone evoked a tenfold increase in EGF in the pancreas, but had no effect on levels in the kidney or liver. In contrast, T₃ evoked a sixfold increase in the EGF level in the kidney, but had no effect on levels in the pancreas or liver. Hormonal administration had no effect on EGF levels in the stomach. Within the intestinal tract, cortisone had no effect on the luminal EGF content of the duodenum, jejunum or ileum, but caused a decrease in the midjejunum. T₃ evoked a decrease in the luminal EGF content of the ileum. The effect of cortisone on mucosal EGF content varied between regions; an increase was seen in the duodenum with a decrease in the midjejunum and ileum. T₃ administration resulted in a significant decrease in EGF only in the mucosa of the ileum. The EGF-degradative capacity of the luminal contents of the jejunum and ileum, as studied in vitro, were increased by treatment with both T₃ and cortisone. However, no direct correlation was observed between alterations of the EGF content in the lumen or mucosa and the increased degradative capacity. We therefore conclude that modulation of EGF levels within the intestine cannot be explained exclusively by alterations in the degradation rates. The results indicate that both adrenal and thyroid glands may play an important role in the modulation of EGF levels in the developing rat.

Journal of Endocrinology (1991) 131, 95–100

SUMMARY

Sera from inbred rabbit strains have been studied to determine radio-thyroxine distribution after electrophoresis, to measure protein binding capacity for thyroxine (T₄) and to examine possible interrelationships of binding capacity and serum protein-bound iodine (PBI). In electrophoretic studies at pH 7·4, serum albumin and prealbumin, the latter a previously unrecognized carrier of T₄ in the rabbit, are the principal transport proteins. At pH 9·0, prealbumin is the major carrier (61% of tracer). Prealbumin also binds significant quantities of tri-iodothyronine. The mean binding capacity of serum prealbumin for T₄ in 12 rabbit strains was 500 μg/100 ml.

Protein-bound iodine levels are known to be strain-dependent in the rabbit. In the current studies, however, there was no strain-specificity of prealbumin binding capacity, and no correlation between PBI and binding capacity of prealbumin, the principal T₄-specific transport protein. These observations suggest that factors other than the capacity of binding proteins for T₄ may be primary determinants of hormone levels (PBI) in blood in the rabbit.

Species differences have been described in the thyroxine-serum protein complexes of various mammals (Blumberg & Robbins, 1960; Farer, Robbins, Blumberg & Rail, 1962; Rösler, 1967a, b). The thyroid hormone-binding serum proteins of the pouched mammals have not been studied. Because this order (Marsupialia) has had a long separate evolutionary history, its protein patterns are of interest and have been used to study speciation (Kirsch & Poole, 1967). We report here electrophoretic studies of the binding of thyroid hormones by serum proteins of the grey kangaroo.

Sera were obtained from Eastern (Macropus giganteus) and Western (Macropus fuliginosus) grey kangaroos and from a hybrid (Eastern x Western) maintained by the Division of Wildlife Research, C.S.I.R.O. Except for one Western specimen, all sera were from adult females. Sera were labelled with 0·9–2·4 μg. [¹³¹I]thyroxine (T₄)/100 ml. or 0·9–1·4 μg. [¹²⁵I]tri-iodothyronine (T₃)/100 ml. obtained from Abbott Laboratories (North Chicago,

SUMMARY

[4-¹⁴C]Testosterone, [4-¹⁴C]androstenedione and [4-¹⁴C]oestrone were incubated with testicular tissue obtained from an 18-yr.-old patient with the complete form of the testicular feminization syndrome.

Considerable biosynthesis of testosterone from androstenedione occurred, but metabolism of testosterone by the tissue was minimal. The small phenolic fraction from these incubations did not contain any recognizable oestrogens.

Metabolism of oestrone was almost complete, less than 4% being recovered as unchanged oestrone. Of eight areas of radioactivity found during chromatography, five were shown to be associated with oestrone, 2-methoxyoestrone, oestradiol-17β, 2-hydroxyoestradiol-17β, and 2-methoxy-oestradiol-17β. Chromatographic evidence suggested that an oxo-oestrone and a compound more polar than oestriol were present. No oestriol was found.

The results confirm those of other workers to the effect that testosterone is the major metabolite of androstenedione in feminizing testes. Incubation of the testes with oestrone showed them also to possess the enzyme systems necessary for hydroxylation at position 2 and the subsequent methylation of this group.

Urinary steroid measurements before and after removal of the testes showed these organs to be actively secreting.

Attempts to demonstrate oestrogenic activity in the urine additional to that accounted for by chemical estimation were unsuccessful.

ABSTRACT

The effect of immunizing against somatostatin (SRIF), with SRIF conjugated to bovine thyroglobulin, was examined in cross-bred sheep fed either cut pasture or lucerne pellets. Plasma concentrations of GH were unaffected by SRIF immunization, but were lower in pellet-fed sheep. Plasma concentrations of insulin-like growth factor I (IGF-I) increased after immunization in sheep on both diets. Pasture-fed sheep had lower plasma concentrations of IGF-I than those on pellets. Sheep showed a small increase in growth rate in response to immunization. Immunization had no effect on carcass composition and did not affect plasma concentrations of IGF-II, free fatty acids or glucose. The results show that even though SRIF immunization increases plasma concentrations of IGF-I, it does not necessarily result in a large increase in growth rate.

J. Endocr. (1987) 112, 27–31

ABSTRACT

Competitive tracer binding studies using radio-iodinated insulin-like growth factor-I and -II (¹²⁵I-labelled IGF-I and ¹²⁵I-labelled IGF-II) together with size exclusion chromatography and IGF-I affinity chromatography have been used to characterize IGF binding protein activity in ovine tissue fluids. Binding proteins of >200, 150 and 40–50 kDa were revealed in these studies and shown to be widely distributed in body fluids. Thus, the > 200 kDa binding protein, which is IGF-II specific, is present in plasma from mature sheep, colostrum and follicular fluid as well as fetal sheep plasma. This may be the ovine equivalent of the soluble type-2 IGF receptor recently identified in rat serum. The presence of a 150 kDa binding protein, of mixed specificity for IGF-I and IGF-II, in fetal and mature sheep plasma was confirmed in these studies. This protein, previously believed to be restricted to vascular fluids, was also identified in mammary lymph, follicular fluid and as a minor component in vitreous humor. Binding proteins of 40–50 kDa were revealed in every fluid tested and multiple variants with distinct specificities were also suggested. This was investigated by IGF-I affinity chromatography using mature sheep plasma. Following passage through the affinity adsorbent, binding of ¹²⁵I-labelled IGF-I to proteins in the 40–50 kDa region was abolished but when ¹²⁵I-labelled IGF-II was used as tracer, a binding protein of 40–50 kDa was still observed.

Thus sheep plasma contains at least two 40-50 kDa binding proteins. The competitive tracer binding studies indicated that one such protein demonstrates mixed specificity for IGF-I and -II while the other strongly favours IGF-II.

Journal of Endocrinology (1989) 120, 429–438

ABSTRACT

The mechanisms of lymphatic-vascular transfer across the ovarian vascular pedicle were studied in anaesthetized sheep 8–15 days after ovulation. [³H]Prostaglandin F_2α (PGF_2α), [¹⁴C]mannitol and [³⁶Cl]Na were infused continuously into either a uterine lymphatic or a uterine vein and the kinetics of transfer into the adjacent utero-ovarian vein or ovarian plasma were studied. Transfer occurred according to the sequence [³⁶Cl] > [¹⁴C] > [³H] indicating that PGF_2α is not transferred by rapid diffusion, as with [³⁶Cl]Na, nor by a paracellular route, as with [¹⁴C]mannitol, but by a slower process probably involving facilitated diffusion.

Transfer into the adjacent utero-ovarian vein or ovarian blood was greater when compounds were infused into a uterine lymphatic than into a uterine vein. Substantially more [³H]PGF_2α occurred in the adjacent corpus luteum than either of the other compounds after a lymphatic infusion. Intra-lymphatic infusion of PGF_2α stimulated the release of ovarian oxytocin but the effect was not confined to the adjacent ovary. Intravenous (jugular) infusion of PGF_2α failed to stimulate ovarian oxytocin secretion whereas close-arterial infusion into the ovaries was effective, and the possibility was investigated that any systemic effect of PGF_2α was mediated through neural mechanisms. Noradrenaline and acetylcholine were both effective in causing the release of ovarian oxytocin when infused close-arterially into the ovary. With infusions of acetylcholine, ovarian oxytocin secretion rate was increased over fivefold without any change in posterior pituitary release. Noradrenaline and acetylcholine produced a concomitant fall in ovarian blood flow, and neurotransmitter-induced ischaemia may have played a role in ovarian oxytocin release. The finding that PGF_2α infused into a uterine lymphatic stimulates ovarian secretion of oxytocin, and that the effect is bilateral whereas PGF_2α accumulation in ovarian tissue is unilateral, implies that its mechanism of action may not be solely directed at the luteal cell.

Journal of Endocrinology (1989) 122, 147–159

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17β-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 ± 3.0 pg/ml), compared with LF mice (5.2 ± 2.3 pg/ml; P≤ 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 ± 3.0 pg/ml) versus the LF group (32.7 ± 8.4 pg/ml; P≤ 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 ± 6.7 ng/ml) versus the 5015 group (60.1 ± 4.9 ng/ml; P≤ 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 ± 0.2 mmol/l) than LF and control mice (0.5 ± 0.1 and 0.6 ± 0.1 mmol/l respectively; P≤ 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 ± 0.1 mmol/l) than LF and control females (0.4 ± 0.1 and 0.6 ± 0.1 mmol/l; P≤ 0.05). Mean vaginal pH of VHF females (6.41 ± 0.09) was lower than 5015 females (6.76 ± 0.10; P≤ 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

ABSTRACT

Plasma and mammary efferent lymph concentrations of insulin-like growth factor I (IGF-I) were determined in lactating ewes before and after treatment with GH (10 mg/day) for 3 days. The lymph:plasma ratio of IGF-I increased from 0·34 to 0·47 after GH treatment when the IGF-I content of plasma increased by 19·4 nmol/l (from 32·1 nmol/l) and lymph by 13·7 nmol/l (from 10·7 nmol/l). This increase in the relative content of IGF-I in lymph was associated with increased lymph content of IGF-I in a lower molecular mass pool (nominally 50 kDa) derived by size exclusion chromatography.

GH treatment increased the total binding capacity for IGF-I in both high (150 kDa) and low (50 kDa) molecular mass pools of plasma and the 150 kDa pool in lymph but there was a proportionally greater increase in 50 kDa total binding in lymph relative to plasma. Further, GH treatment increased the 'saturation' of the 50 kDa binding proteins but decreased the 'saturation' of the 150 kDa fraction, in both plasma and lymph.

Ligand blot analysis of IGF-binding proteins (IGFBPs) in plasma and lymph showed that GH treatment of lactating sheep increased IGFBP-3 and decreased IGFBP-2 in plasma and lymph. Radioimmunoassay of IGFBP-2 showed that while GH treatment reduced the plasma content of IGFBP-2 by about half, the lymph:plasma ratio was increased from 0·68 to 0·87.

GH treatment of lactating ewes not only increased the IGF-I content of plasma but increased the apparent efficiency of transfer of IGF-I across capillary endothelium to mammary efferent lymph.

Journal of Endocrinology (1992) 132, 339–344

SLC30A8 encodes the zinc transporter ZnT8. SLC30A8 haploinsufficiency protects against type 2 diabetes (T2D), suggesting that ZnT8 inhibitors may prevent T2D. We show here that, while adult chow fed Slc30a8 haploinsufficient and knockout (KO) mice have normal glucose tolerance, they are protected against diet-induced obesity (DIO), resulting in improved glucose tolerance. We hypothesize that this protection against DIO may represent one mechanism whereby SLC30A8 haploinsufficiency protects against T2D in humans and that, while SLC30A8 is predominantly expressed in pancreatic islet beta cells, this may involve a role for ZnT8 in extra-pancreatic tissues. Consistent with this latter concept we show in humans, using electronic health record-derived phenotype analyses, that the ‘C’ allele of the non-synonymous rs13266634 SNP, which confers a gain of ZnT8 function, is associated not only with increased T2D risk and blood glucose, but also with increased risk for hemolytic anemia and decreased mean corpuscular hemoglobin (MCH). In Slc30a8 KO mice, MCH was unchanged but reticulocytes, platelets and lymphocytes were elevated. Both young and adult Slc30a8 KO mice exhibit a delayed rise in insulin after glucose injection, but only the former exhibit increased basal insulin clearance and impaired glucose tolerance. Young Slc30a8 KO mice also exhibit elevated pancreatic G6pc2 gene expression, potentially mediated by decreased islet zinc levels. These data indicate that the absence of ZnT8 results in a transient impairment in some aspects of metabolism during development. These observations in humans and mice suggest the potential for negative effects associated with T2D prevention using ZnT8 inhibitors.