Effects of glucocorticoids on circulating levels and hepatic expression of insulin-like growth factor (IGF)-binding proteins and IGF-I in the adrenalectomized streptozotocin-diabetic rat.

Circulating levels and hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) are increased in insulin-deficient streptozotocin (STZ)-diabetic rats. Glucocorticoids stimulate and insulin suppresses hepatocellular expression of IGFBP-1 in vitro. We asked whether increased IGFBP-1 expression in STZ-diabetic animals is due to an effect of insulin deficiency per se or whether insulin deficiency represents a permissive state where glucocorticoids may play an important role in the regulation of IGFBP-1 and other circulating peptides involved in the modulation of IGF bioactivity. Intact female Sprague-Dawley-derived rats and rats undergoing bilateral adrenalectomy (ADNX) were injected with STZ (140 mg/kg) or buffer. Corticosterone acetate (50 mg/kg) or vehicle was administered to diabetic and nondiabetic animals immediately after ADNX and 24 h later. All rats were killed 48 h after surgery and/or STZ administration. Serum [125I]IGF-I-binding activity was increased 4-fold (P < 0.01), and Western ligand and immunoblotting demonstrated that levels of IGFBP-1 were high in intact STZ-diabetic animals. ADNX prevented these effects of STZ-diabetes, and corticosterone treatment restored serum IGF-binding activity and IGFBP-1 to intact diabetic levels. Similarly, Northern analysis demonstrated that the abundance of hepatic IGFBP-1 mRNA was increased 6-fold in intact STZ-diabetic animals (P < 0.01), but not in adrenalectomized diabetic animals. Corticosterone treatment restored hepatic IGFBP-1 mRNA to intact diabetic levels, and serum concentrations of corticosterone correlated with the abundance of IGFBP-1 mRNA (r = 0.475; P < 0.01), indicating that glucocorticoids play an important role in the regulation of expression of IGFBP-1 in insulin-deficient animals. In contrast, neither ADNX nor corticosterone altered the abundance of hepatic IGFBP-1 mRNA levels in nondiabetic animals. This pattern of regulation appeared to be specific; serum levels of immunoreactive IGFBP-2 and -4 tended to rise in adrenalectomized animals, and levels of IGFBP-3 were not affected by either ADNX or corticosterone treatment. Of note, serum levels of IGF-I by RIA were reduced in STZ-diabetic animals compared to control values (168 +/- 16 vs. 587 +/- 55 ng/ml, respectively; P < 0.01), were partially restored toward control values with ADNX (320 +/- 22 ng/ml), and were reduced again by corticosterone treatment (195 +/- 26 ng/ml), indicating that glucocorticoids also contribute to the regulation of IGF-I levels in insulin-deficient animals. The abundance of IGF-I mRNA was reduced in STZ-diabetic animals, and ADNX also partially prevented this effect of diabetes.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid effects on IGF-1/somatomedin-C and somatomedin inhibitor in streptozotocin-diabetic rats.

Diabetes is associated with a fall in serum levels of insulin-like growth factor-1/somatomedin-C (IGF-1/Sm-C) and a rise in somatomedin inhibitor, a factor which antagonizes somatomedin action. We attempted to determine if the presence of glucocorticoids was required for diabetes-related alterations in these circulating growth factors. Diabetes was induced with streptozotocin in intact or adrenalectomized rats. Adrenalectomized-nondiabetic and adrenalectomized-diabetic rats were given either no glucocorticoids or daily hydrocortisone acetate at 0.5 or 50 mg/kg body weight, and killed 48 hours after streptozotocin treatment. After serum fractionation via size exclusion high performance liquid chromatography (HPLC), IGF-1/Sm-C was determined by radioimmunoassay, and somatomedin inhibitor by bioassay according to the ability of serum fractions to blunt cartilage stimulation by normal serum. Intact-diabetic rats had 22% weight loss, glucose 427 mg/dL, and beta-hydroxybutyrate 7.2 mmol/L (all P less than .001 v control). Serum IGF-1/Sm-C levels in intact-diabetic rats were decreased 71%, while somatomedin inhibitor rose to 470% of the control values (both P less than .004). Adrenalectomized-diabetic rats displayed comparable hyperglycemia (greater than 400 mg/dL) and decline in IGF-1/SmC, with or without glucocorticoid replacement. However, adrenalectomized-diabetic rats had greatly reduced weight loss (10%), beta-hydroxybutyrate (1.5 mmol/L), and somatomedin inhibitor (59% of control), all P less than .01 v intact-diabetic. Hydrocortisone 0.5 mg/kg in these animals increased weight loss but had no significant effect on beta-hydroxybutyrate or somatomedin inhibitor levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The increase in hepatic tyrosine aminotransferase activity by ethanol administration involves an acceleration of enzyme synthesis.

The present study was conducted to examine the nature of the increase in tyrosine aminotransferase (TAT) activity by acute ethanol administration. A significant rise in aminotransferase activity was observed as early as 1 hr after intact rats were gavaged with ethanol. Ethanol administration also increased TAT activity in adrenalectomized rats. Inhibition of ethanol metabolism by pyrazole administration had no effect on the ethanol-induced increase in TAT activity. Immunochemical analyses revealed that the enhancement of TAT activity in ethanol-fed rats correlated with an increase in aminotransferase protein. Measurement of the rate of TAT synthesis showed that in ethanol-fed rats, [3H]leucine was incorporated into the aminotransferase protein at a higher rate than in controls by a factor which was similar to the enhancement in enzyme activity. Our findings indicate that an acceleration of TAT synthesis fully accounts for the increase in TAT activity during the early stage of enzyme induction. TAT induction by ethanol administration is not dependent upon an increase in adrenal corticosteroid production, nor does it require ethanol metabolism.

The binding of FITC-insulin to ANAE-positive cells in rat thymus.

To determine if thymic macrophages have insulin receptors, alternate sections of rat thymus were stained with FITC-insulin and examined for nonspecific esterase (ANAE) activity. Cells showing a diffuse ANAE staining pattern also bound FITC-insulin. These cells were concentrated in the cortico-medullary border and increased in number following administration of cortisol. Thymic macrophages may be insulin-dependent and therefore could be malfunctional in diabetes.

Suppressed proliferation of lymphatic tissue in diabetic and adrenalectomized-diabetic rats.

One week following induction of diabetes in rats by alloxan administration, the thymus and spleen showed marked involution and a highly significant depression of in vivo incorporation of [3H]deoxycytidine into the DNA of these tissues. Adrenalectomy of diabetic rats reduced the decline of lymphatic tissue weight, but uptake of [3H]deoxycytidine into thymic and spleen DNA remained suppressed. These results indicate that in alloxan-induced diabetes, the observed depressed lymphatic tissue weight and cellular proliferation are not simply due to the elevated plasma level of corticosteroids, but are either a result of the insulin deficiency per se or due to other metabolic alterations associated with the diabetic condition.

An inhibitory effect of sulfhydryl compounds in the assay of rat and mouse thymic terminal deoxynucleotidyl transferase (TdT).

Terminal deoxynucleotidyl transferase (TdT) was assayed in 100,000 X g supernatants of homogenized thymus using 3H-dGTP and an oligo p(dA)12-18 primer. 2-Mercaptoethanol (2-ME) caused a depression of activity with rat and mouse thymus extracts but increased activity using bovine or lamb thymus extracts. Glutathione (GSH), L-cysteine and dithiothreitol (DTT) also showed an inhibitory effect with the rat thymus extract. Inhibition was significant at concentrations of sulfhydryl compounds commonly included in TdT assays (1 mM-2 mM).

In vitro insulin-stimulated conversion of [U-14C]glucose to 14CO2 by rat thymocytes.

Addition of bovine insulin to thymocytes from adrenalectomized rats resulted in stimulation of [U-14C]glucose conversion to 14CO2. A significant enhancement of 14CO2 formation by insulin occurred by 30 min of incubation, and was consistently observed at an insulin concentration of 10(-8) M. The response to insulin was similar at 0.55 and 1.1 mM glucose, and was obtained at three cell concentrations (0.5, 1.0, 2.0 X 10(8) cells/ml). The incorporation of [3H]leucine into trichloroacetic acid-precipitable material was significantly increased by 10(-6) and 10(-8) M insulin. Cycloheximide, at a level of 2.5 X 10(-5) M, suppressed [3H]leucine incorporation by 93% and inhibited the stimulation of 14CO2 formation by insulin. We conclude that insulin can enhance the formation of 14CO2 from [U-14C]glucose by thymocytes in vitro, and that this response may require the synthesis of one or more proteins.

A differential effect of L-serine on the incorporation of 3H-deoxycytidine and 3H-thymidine into DNA of rat thymocytes.

The addition of L-serine to short-term cultures of rat thymocytes stimulated the incorporation of 3H-deoxycytidine into DNA, but simultaneously depressed the incorporation of 3H-thymidine into DNA.