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ABSTRACT

Diabetes-induced growth retardation in the rodent is associated with both reduced circulating insulin-like growth factor-I (IGF-I) and enhanced levels of inhibitors of somatomedin activity. IGF-binding proteins (IGFBPs) are present in the circulation and tissue fluids and are believed to modulate the actions of IGF-I. Since elevated concentrations of the IGFBPs may contribute to the enhanced somatomedin-inhibitor activity observed in serum from diabetic animals, we have examined the amounts of hepatic IGFBP-1, -2, -3 and -4 mRNA in the spontaneously diabetic BioBreeding/Worcester rat.

The study used two types of diabetic animal: mildly diabetic animals, which received suboptimal insulin treatment (0.5–1 U/day) and diabetic animals, which received intensive insulin treatment (3–6 U/day). A significant increase in the amount of IGFBP-1 and IGFBP-2 mRNA was seen 1 month and 3 months after the onset of diabetes. Intensive insulin treatment for 3 weeks normalized the amount of IGFBP-1 mRNA in diabetic rats and resulted in a decrease in IGFBP-2 mRNA. In contrast to the increase in IGFBP-1 and IGFBP-2 mRNA, a significant decrease in IGFBP-3 mRNA was seen in diabetic rats (54.6% of control, P < 0.0005 and 64.6% of control, P < 0.005 for 1 and 3 months respectively) and intensive insulin treatment for 3 weeks did not restore the IGFBP-3 mRNA level in diabetic rats. No significant difference in IGFBP-4 mRNA levels was seen in diabetic compared with non-diabetic rats. When serum was analysed by ligand blotting the major finding was a reduction in the 39–42kDa binding protein. No increase in 29–30kDa IGFBP in the serum was detected in the diabetic rats. From these studies we conclude that the major change in IGFBPs in mildly hyperglycaemic spontaneously diabetic rats is a decrease in IGFBP-3. The changes in hepatic IGFBP-1 and -2 mRNA do not appear to be of sufficient magnitude to result in an increase in serum concentrations of these binding proteins.

ABSTRACT

Previous studies have established that the IGFs are involved in oestrogen-induced uterine proliferation. IGF-binding proteins (IGFBPs) are present in most biological fluids and tissues and may modulate the actions of the IGFs. We examined uterine, hepatic and renal expression of the IGFBPs throughout the oestrous cycle and investigated the effects of oestradiol (OE₂) on IGFBP expression in ovariectomized (ovx) rats. Uterine expression of IGFBPs-1 and -3 showed a definite variation throughout the oestrous cycle with highest levels during dioestrus. In the liver and kidney the changes in IGFBP-1 and IGFBP-3 mRNA abundance were the opposite of those observed in the uterus, with the highest levels observed during oestrus. Administration of OE₂ to ovx rats decreased uterine IGFBP-3 mRNA and increased IGFBP-4 mRNA levels. In these rats there were no consistent changes in renal IGFBP-1 or IGFBP-3 mRNAs; however, a significant increase in IGFBP-4 mRNA was observed in this tissue, as in the uterus. In the liver an increase in IGFBP-1 mRNA and a decrease in IGFBP-3 mRNA levels were observed in rats treated with OE₂. Despite changes in uterine, hepatic and renal IGFBP mRNA levels, no significant variation was seen in serum IGFBPs as determined by ligand blotting of sera. These data demonstrate that there is a cyclical variation in the expression of the IGFBPs in the uterus, kidney and liver, and that OE₂ is able to modulate differentially IGFBP expression in these tissues.

ABSTRACT

The testicular feminized (Tfm) mouse lacks functional androgen receptors and develops with a female external phenotype and internal testes. The testes of these animals contain normal, or close to normal, numbers of Leydig cells but secrete very low amounts of androgen due to a lack of 17α-hydroxylase activity. To determine whether this loss of activity is due to a lack of enzyme synthesis or a change in catalytic activity we have examined 17α-hydroxylase cytochrome P-450 (P-450_17α) protein and mRNA levels in the testes of Tfm mice.

Levels of P-450_17α protein were measured by immunoblotting, while mRNA was measured following reverse transcription (RT) and amplification by the polymerase chain reaction (PCR). Conditions for RT-PCR were determined which allowed semiquantification of P-450_17α mRNA relative to β-actin mRNA. In extracts of Tfm testes P-450_17α protein was undetectable using antiserum against porcine P-450_17α. In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-450_17α mRNA was detectable in both control and [ill] testes but, expressed in terms of β-actin mRNA, levels of P-450_17α mRNA in control testes were 40-fold higher than those in [ill] testes. If the total amount of RNA extracted from each testis is taken into account then P-450_17α mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17α-hydroxylase activity in [ill] testes is related to reduced protein synthesis. Previous results have shown that androgens reduce P-450_17α mRNA levels in cultured Leydig cells. Results from this study suggest, however, that androgens are required to induce normal levels of P-450_17α mRNA in Leydig cells.

ABSTRACT

Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mm ZnCl₂. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P<0·05, P<0·005 and P<0·01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis.

Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with ¹²⁵I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P<0·05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.

ABSTRACT

It is well established that oestrogens can stimulate prolactin (PRL) secretion as well as the expression of the vasoactive intestinal peptide (VIP) gene whose product is also a potent PRL secretagogue. Previous evidence has supported both an autocrine and a paracrine role for pituitary VIP in PRL release in vitro; however, the cellular origin of VIP in pituitary tissue still remains poorly defined. In these studies, we have demonstrated by in situ hybridisation that VIP RNA is detected in the anterior pituitaries of chronically hyperoestrogenised rats, but not in those of untreated animals. Using a double-probe labelling procedure, VIP RNA has been shown to be present in a subpopulation of PRL-producing cells, while colocalisation of VIP and GH RNA was not observed. VIP gene expression in the rat anterior pituitary gland was characterised by the presence of two alternatively polyadenylated transcripts, 1·7 kb and 1·0 kb in size. We have generated a probe specific for the 1·7 kb transcript and double-labelling studies also showed definitive colocalisation with PRL mRNA. Our results demonstrating the presence of VIP RNA in PRL-producing cells thus suggest that VIP may play an autocrine role in PRL hypersecretion under conditions of oestrogen-induced hyperplasia.