Experimental confounding factors affecting stability, transport and metabolism of flavanols and hydroxycinnamic acids in Caco-2 cells.

Studying bioavailability of polyphenols is essential to understand the health effects of these compounds. Human epithelial cells are commonly used in intestinal absorption and transport experiments but the changes polyphenols undergo during incubation, due to their chemical instability under the cell culture conditions, are scarcely known and might lead to inaccurate conclusions. Based on abundance of flavanols and hydroxycinnamic acids in the diet, epicatechin, epicatechin-3-gallate and procyanidin B2 as flavanols along with 5-caffeoylquinic and 3,5-dicaffeoylquinic acids as hydroxycinnamic acids were selected to comparatively evaluate their absorption and metabolism using an in vitro Caco-2 cell model. Special emphasis was paid to the structure-stability relationship of these phenolic compounds in Dulbecco's Modified Eagle's Medium (DMEM) under the cell culture conditions. The tested compounds were scarcely absorbed and minimally metabolized by the intestinal epithelium cells. The cell transport study showed prevalent efflux for flavanols opposite to absorption for hydroxycinnamates. Intestinal metabolism revealed that hydroxycinnamates were preferentially hydrolyzed and subsequently methylated, whereas hydrolysis of flavanols could not be confirmed, being mostly conjugated to sulfate, methyl- and methyl-sulfate derivatives. It is noteworthy that methyl derivatives of procyanidin-B2 were detected inside Caco-2 cells, confirming its absorption. In addition, culture medium influenced phenol isomerization to a higher extent than cells. In conclusion, hydroxycinnamates were better absorbed than flavanols although their bioavailability was limited in this intestinal cell model.

Glucagon-like peptide-1 improves beta-cell antioxidant capacity via extracellular regulated kinases pathway and Nrf2 translocation.

Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation.

A phenolic extract from grape by-products and its main hydroxybenzoic acids protect Caco-2 cells against pro-oxidant induced toxicity.

Grape/wine industry produces large amounts of by-products, however knowledge on their health-promoting qualities is limited. This study investigated the effects of a grape phenolic extract (GPE) and its phenolic compounds, gallic acid (GA) and syringic acid (SA) on human intestinal Caco-2 cells, directly or after cytotoxicity induced by tert-butylhydroperoxide (t-BOOH). Direct treatment with 0.1-10 µg/mL GPE, or 0.1-10 µM GA and SA produced no major cytotoxic effect, either changes in antioxidant defences (glutathione content, glutathione peroxidase and reductase activities) or protein damage (carbonyl groups). However, 10 µg/mL GPE, 1 and 10 µM GA and 10 µM SA decreased reactive oxygen species (ROS) production. Pre-treatment with GPE, SA and GA at the same concentrations for 20 h showed that 10 µg/mL GPE and 10 µM GA or SA significantly counteracted ROS increase induced by t-BOOH. 10 µg/mL GPE and 1-10 µM GA or 10 µM of SA significantly reduced pro-oxidant-induced cytotoxicity. 1-10 µg/mL GPE, 1-10 µM GA and 10 µM SA significantly recovered both depleted glutathione and enhanced glutathione reductase and peroxidase activities, and reduced protein oxidative damage. Therefore, treatment with realistic concentrations of GPE and its main hydroxybenzoic acids protected Caco-2 cells against induced oxidative stress.

Protein tyrosine phosphatase 1B modulates GSK3ß/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity.

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B(-/-) hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)ß/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B(-/-) cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B(-/-) mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAP-induced liver failure.

[Characterization of cocoa fiber and its effect on the antioxidant capacity of serum in rats]. / Caracterización de la fibra de cacao y su efecto sobre la capacidad antioxidante en suero de animales de experimentación.

OBJECTIVES: The aim of this study was to characterize the physico-chemical properties of cocoa fibre (CF), to analyze its polyphenolic content and antioxidant capacity in vitro, and to investigate the effect of the administration of a polyphenolic extract of this cocoa fiber on the antioxidant capacity of the serum in rats. METHODS AND MATERIALS: Dietary fiber (DF) composition and polyphenolic (PP) content of the cocoa fiber were analyzed. The antioxidant capacity of the CF was determined by means of its reduction power (FRAP) and the capacity to scavenge free radicals (ABTS). To evaluate the bioavailability and the antioxidant capacity in vivo of the phenolic compounds of CF, an extract of these compounds was administred in the stomach of the rats with a gastric probe (100 mg PP/kg), taking blood samples at different time intervals. Sera were analyzed by HPLC to determine the presence/absence of PP or PP-metabolites. In orther to evaluate the antioxidant capacity of the serum FRAP and ABTS methods were used. RESULTS: Cocoa fiber was an excellent source of DF, with a high content of total dietary fiber (TDF), over 60% of the dry matter, made up mainly of insoluble dietary fiber (IDF; 83% of TDF). This fiber had just 1.15% of polyphenols, with low antioxidant activity. After intragastric administration of the PP-rich fraction a fast and measurable absorption of the CF polyphenols was observed, being epicatechin the main PP in blood. The absorption of this PP confers a significant, although transitory increase of the serum antioxidant capacity 10-45 minutes post-gavage; after this time, the antioxidant capacity progressively decreased reaching basal levels after 6 h. CONCLUSIONS: Cocoa fiber can be considered as an excellent source of DF, mainly insoluble dietary fiber; therefore, it could be used as an ingredient in fiber-rich functional foods. Besides the benefits derived from its high fiber content, the CF would provide protection against oxidative damage by means of its content in phenolic compounds (epicatechin) wich are absorbed maintaining the antioxidant properties in vivo.

Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation.

Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.

Protein-caloric food restriction affects insulin-like growth factor system in fetal Wistar rat.

We have previously shown that fetuses from protein-caloric undernourished pregnant rats (35% of control diet during the last week of pregnancy) at 21.5 d post coitum exhibit increased beta-cell mass. This alteration is correlated with increased insulinemia and total pancreatic insulin content, a pattern similar to that reported in infants of mild diabetic mothers. In this work, we investigated in undernourished fetuses: 1) whether availability of growth factors such as insulin, GH, and IGFs and their binding proteins (IGFBPs) could be implicated in this alteration, and 2) the beta-cell mitogenic response to IGFs in vitro. The results show that maternal undernutrition increases pancreatic IGF-I expression and islet IGF-I receptor content in undernourished fetuses, whereas hepatic IGF-I expression and serum IGF-I levels were decreased. No changes were observed in serum IGF-II, and its expression was diminished in undernourished pancreases and unchanged in the liver, compared with control fetuses. Serum levels and liver and pancreatic mRNA expression of IGFBP-1 were found to be normal in undernourished fetuses, whereas the serum concentration and abundance of IGFBP-2 mRNA in pancreas were increased. Finally, the beta-cell mitogenic response to IGFs in vitro was significantly increased in undernourished fetal islets, compared with controls. In conclusion, in undernourished fetuses the increased beta-cell mass can be related to the stimulation of replicative beta-cell response due to locally increased pancreatic IGF-I mRNA; this effect is perhaps potentiated or favored by the enhanced islet IGF-I receptor content and pancreatic IGFBP-2 gene expression.

Age-dependent adaptation of the liver thyroid status and recovery of serum levels and hepatic insulin-like growth factor-I expression in neonatal and adult diabetic rats.

The effect of treatment with thyroxine (T(4)) on the hepatic deiodinase (5'D-I) activity and triiodothyronine (T(3)) content and on insulin-like growth factor-I (IGF-I) secretion and mRNA hepatic expression were studied in neonatal and adult diabetic (D) rats and compared with 4 thyroidectomized (Tx) groups: neonatal and adult Tx rats treated or not with T(4). Serum T(3) and T(4) decreased by 92% in both Tx populations and by 80% to 70% in D adults according to the severity of diabetes: -70 mg/kg body weight (BW) (D(70)) or 50 mg/kg BW (D(50)) of streptozotocin (STZ) injected, whereas only a 30% to 33% decrease was found in D neonates. A similar decrease of liver 5'D-I activity and T(3) concentrations was found in neonatal and adult Tx rats, whereas a significant reduction in those parameters was observed only in adult diabetics, either D(70) or D(50), but not in D neonates. Serum levels and liver mRNA expression of IGF-I determined by ribonuclease protection assay, plasma and pituitary growth hormone (GH), plasma insulin, and glycemia were also measured in both D populations. A decrease in circulating IGF-I, previously reported for Tx adult rats, was also found in both D populations. T(4) treatment recovered IGF-I and liver T(3) in both Tx groups and D neonates, but not in D adults. These results show an age-dependent adaptation of the liver thyroid economy in diabetes, as hepatic 5'D-I does not respond to diabetes in neonates and IGF-I is insensitive to T(4) treatment in adult diabetics and suggest a positive correlation between hepatic T(3) content and IGF-I expression in conditions of diabetes and Tx.

Effects of early undernutrition on the brain insulin-like growth factor-I system.

Undernutrition reduces circulating concentrations of insulin-like growth factor (IGF)-I, but how it affects the brain IGF system, especially during development, is largely unknown. We have studied IGF-I, IGF-II, IGF receptor and IGF binding protein (BP)-2 mRNA expression in the hypothalamus, cerebellum and cerebral cortex of neonatal rats that were food restricted beginning on gestational day 16. One group was refed starting on postnatal day 14. Rats were killed on postnatal day 8 or 22. Undernutrition did not produce an overall reduction in brain weight at either age but, at 22 days, both the cerebellum and hypothalamus weighed significantly less. At 8 days, no change was detected in the central IGF axis in response to undernutrition. However, in 22-day-old undernourished rats, IGF-I and IGF receptor mRNA expression were increased in both the hypothalamus and cerebellum, while IGFBP-2 was decreased, but only in the hypothalamus. Refeeding had no effect on any of these parameters. These results suggest that the hypothalamus and cerebellum respond to malnutrition and the decrease in circulating IGF-I, a peptide fundamental for growth and development, by increasing the local production of both the growth factor and its receptor in attempt to maintain normal development.

Influence of hypothyroidism on circulating concentrations and liver expression of IGF-binding proteins mRNA from neonatal and adult rats.

The aim of this work was to study the influence of the endocrine balance between thyroid hormones, insulin and growth hormone (GH) on the regulation of insulin-like growth factor binding proteins (IGFBPs), complementing a study previously reported for insulin-like growth factors (IGFs) in similar populations. Serum concentrations of IGFBPs-1 to -3 were assayed by Western ligand blot and their mRNA expression in the liver assayed by RNase protection assay in the hypothyroid populations: thyroidectomized and mercapto-1-methylimidazole (MMI)-treated neonates, and thyroidectomized adult rats at different periods after thyroidectomy. Serum concentrations of insulin, GH and IGF-I were increased in thyroidectomized neonates and decreased in the other populations. IGFBPs-1 and -2 increased 79% and 50% respectively in thyroidectomized neonatal rats compared with control at 15 days after thyroidectomy, whereas only IGFBP-2 increased (87%) in MMI-treated neonates, which had low serum insulin and GH compared with control on the same days. In thyroidectomized adult rats, IGFBPs-1 and -2 decreased 60% compared with controls on all days studied. Furthermore, when streptozotocin was administered to thyroidectomized neonates and insulin was given to thyroidectomized adult rats to restore insulin to control values in both groups, a differential regulation was found for IGFBPs-1 and -2. The transcriptionally induced decrease in IGFBP-3 (20-25% compared with control in neonates and 50% in adult rats), however, seemed to be regulated by GH and IGF-I. The similarity of changes in IGFBPs found in hypothyroid, undernourished and streptozotocin-induced diabetic neonatal rats suggests that the regulatory effect of insulin or GH on the IGFBPs requires the reduced biologically active thyroid hormone that is found in these three populations.

Regulation of IGF-I and -II by insulin in primary cultures of fetal rat hepatocytes.

During perinatal development, insulin and nutrients, rather than GH, regulate the IGF system. A selective primary culture of fetal rat hepatocytes has been established in our laboratory to elucidate the molecular mechanism of action of the above regulatory factors on IGF-I and -II gene expression during the late fetal period of the rat. In this model we have previously reported a regulatory role for glucose on IGF-I and -II synthesis and secretion. In the same experimental model, we now report that doses of insulin (0.1-5 microM) within the physiological range in rat fetuses during the last stages of gestation evoke an increase of IGF-I and -II mRNA abundance. Insulin regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the hormone. Finally, the insulin-induced rise in IGF-I and -II mRNA was not mediated by stimulation of gene transcription but by increased transcript stability. The results support the hypothesis that insulin plays a major role in IGF regulation at immature stages of development.

Different role of insulin in GLUT-1 and -4 regulation in heart and skeletal muscle during perinatal hypothyroidism.

Two groups of hypothyroid rats were used; one group was given 2-mercapto-1-methylimidazole (MMI) treatment in the drinking water of the mothers and was killed at 2 and 4 days of life, and the other group was given similar MMI treatment and then was thyroidectomized at 5 days of life and killed at 8 or 20 days. Serum insulin, growth hormone (GH), and insulin-like growth factor I (IGF-I) were decreased in MMI-treated rats but increased in MMI-treated plus thyroidectomized rats. No significant reduction of thyroid hormones was observed in 2-day-old MMI rats. Protein and mRNA expression of GLUT-1 increased, and those of GLUT-4 decreased, in the heart in all populations independent of changes in insulin, GH, and IGF-I levels. However, GLUT-4 protein and mRNA expression in quadriceps and gastrocnemius skeletal muscles decreased at 4 days and increased at 8 and 20 days of life in parallel with insulin, GH, and IGF-I levels. GLUT-1 in the skeletal muscles seemed regulated posttranscriptionally and presented a decrease of mRNA expression in all stages studied. A differential sensitivity to insulin regulation of GLUT-1 and GLUT-4 glucose transporters seems to be one of the causes for the tissue-specific regulation of these glucose transporters in heart and skeletal muscles during the perinatal period.

Glucose uptake and glucose transporter proteins in skeletal muscle from undernourished rats.

Undernutrition in rats impairs secretion of insulin but maintains glucose normotolerance, because muscle tissue presents an increased insulin-induced glucose uptake. We studied glucose transporters in gastrocnemius muscles from food-restricted and control anesthetized rats under basal and euglycemic hyperinsulinemic conditions. Muscle membranes were prepared by subcellular fractionation in sucrose gradients. Insulin-induced glucose uptake, estimated by a 2-deoxyglucose technique, was increased 4- and 12-fold in control and food-restricted rats, respectively. Muscle insulin receptor was increased, but phosphotyrosine-associated phosphatidylinositol 3-kinase activity stimulated by insulin was lower in undernourished rats, whereas insulin receptor substrate-1 content remained unaltered. The main glucose transporter in the muscle, GLUT-4, was severely reduced albeit more efficiently translocated in response to insulin in food-deprived rats. GLUT-1, GLUT-3, and GLUT-5, minor isoforms in skeletal muscle, were found increased in food-deprived rats. The rise in these minor glucose carriers, as well as the improvement in GLUT-4 recruitment, is probably insufficient to account for the insulin-induced increase in the uptake of glucose in undernourished rats, thereby suggesting possible changes in other steps required for glucose metabolism.

Effect of thyroxine administration on the IGF/IGF binding protein system in neonatal and adult thyroidectomized rats.

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.

Spray-dried powders as nasal absorption enhancers of cyanocobalamin.

The aim of this work is to describe and characterize a new spray-drying procedure for the production of nasal powders as an alternative to the conventional freeze-drying method. Cyanocobalamin was chosen as the active ingredient and loaded into five different nonsoluble vehicles with high water absorption ability. Then these hydrated particles were suspended in methylene chloride and spray-dried. Particle size, morphology, true, bulk and tapped density, percentage of compressibility, moisture content, water intake, and drug diffusion were studied and significant differences were obtained depending on the nature of the vehicle. The drying method, either the new spray- or the conventional freeze-drying, was less important. Interestingly, an inverse correlation was found between water uptake and drug diffusion. Microcrystalline cellulose, dextran microspheres, and crospovidone were chosen for an in vivo bioavailability study in rabbits. Three other nasal reference formulations and an intravenous solution were also administered. The spray-dried powders showed higher bioavailability than the three nasal reference formulations. The highest absorption enhancement was observed with cellulose microcrystalline powders, which provided a 25% mean absolute bioavailability, followed by crospovidone and dextran microspheres formulations with mean bioavailability values of 14% and 7%, respectively. In conclusion, the new spray-drying method is useful for the production of cyanocobalamin nasal powders.

Influence of type II 5' deiodinase on TSH content in diabetic rats.

The influence of hypothalamic and pituitary type II 5'deiodinase (5'D-II) activities and T3 content on pituitary TSH content was investigated in streptozotocin (STZ)-induced diabetic rats (D). The results show, first, that hypothalamic and pituitary 5'D-II activities were lower in neonatal D rats versus control (C) rats, and the normal developmental pattern was altered. Secondly, when D and C rats were thyroidectomized (Tx) at 25 days of age (D+Tx, C+Tx), pituitary and hypothalamic 5'D-II activities increased ten days later in both populations vs. intact rats, but the percentage of increase was smaller in D+Tx than in C+Tx. The hypothalamic T3 to T4 ratios were also decreased in D+Tx animals (0.38) as compared to C+Tx rats (1.64). The hypothalamic T3 content was reduced by 30% in D as compared to C rats and by 80% in D+Tx as compared to C+Tx rats, showing a defect in hypothalamic T4 deiodination. Pituitary TSH content increased after Tx in D+Tx, but not in C+Tx. These results in diabetic rats indicate that the hypothalamic and pituitary 5'D-II activity and hypothalamic T3 content are affected by diabetes and play a role in the regulation of pituitary TSH content.

Effects of experimental diabetes on renal IGF/IGFBP system during neonatal period in the rat.

Changes in the renal synthesis and concentration of insulin-like growth factors (IGFs) and their serum-binding proteins (IGFBPs) reported in insulin-deficient diabetes mellitus may be implicated in the alterations of the kidney function and morphology accompanying this disease. Most research on this subject has been carried out in adult animals, as well as in peripubertal rats, when the regulation of the IGF system is fully dependent on serum growth hormone (GH). However, relevant differences in the regulatory pathways of IGFs between adult and neonatal periods have been described. To examine the response of the IGF/IGFBP system of neonatal kidney to diabetes, renal IGF-I and -II and IGFBP-1, -2, and -3 concentration and mRNA expression were determined in streptozotocin-induced diabetic rat neonates. Diabetic neonates exhibited a kidney weight-to-body weight ratio higher than that of control rats, together with decreased IGF-I and increased IGF-II renal concentration. Because kidney mRNA expression of both IGFs decreased, the elevated renal IGF-II might result from increased uptake from circulation. Insulin treatment recovered the altered IGFs to control values, indicating the insulin-dependent regulation of IGFs in the neonatal kidney. Elevated levels of the IGFBP-1 and -2 in the kidney of diabetic neonates did not result from changes in their kidney mRNA transcript expression, suggesting also a possible uptake from circulation.

Regulation of insulin-like growth factor-I and -II by glucose in primary cultures of fetal rat hepatocytes.

A selective primary culture of fetal rat hepatocytes was established in our laboratory in order to elucidate the molecular mechanisms of action of different factors and conditions on insulin-like growth factor (IGF)-I and -II gene expression during the perinatal period of the rat. In this model we report that, in a serum-free condition and the presence of non-stimulatory doses of insulin, 5-20 mM glucose evoked an increase of IGF-I and -II mRNA abundance. Glucose regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the carbohydrate. The experiment with 2-deoxyglucose suggests that glucose 6-phosphate, but not its further metabolism, is necessary for the induction of IGF transcript abundance in cultured fetal hepatocytes. Finally, the glucose-induced rise in IGF-II mRNA, the main IGF in fetal stages, was mediated by stimulation of gene transcription and increased transcript stability. The results support the idea that IGFs belong to a family of genes that are positively regulated by glucose.

Liver mRNA expression of IGF-I and IGFBPs in adult undernourished diabetic rats.

To investigate the role played by factors other than GH, such as nutrients and insulin, on IGF-I secretion, adult male rats of 200 g.b.w. were food-restricted for 7 days and then made diabetic by streptozotocin administration (UD). Different groups of UD rats were submitted to the following four day treatments: left untreated (UD), refed (UD+R), treated with insulin (UD+I), or a combination of both refeeding and insulin (UD+R+I). Serum concentration of IGF-I and liver mRNA expression of IGF-I, IGF-binding proteins and GH receptor were measured. Insulin treatment alone partially recovered liver IGF-I and IGFBPs mRNA expression, while refeeding alone had no effect. Only a combination of both insulin and refeeding recovered both parameters. Contrary to the results obtained with a longer period of recovery, these experiments show that serum and mRNA expression of IGF-I and IGFBPs in adult undernourished diabetic rats can be restored by insulin and nutrients administration with no prior restoration of serum and pituitary GH to control values and no compensatory changes in GH receptor gene expression.

Mechanism of hypothyroidism action on insulin-like growth factor-I and -II from neonatal to adult rats: insulin mediates thyroid hormone effects in the neonatal period.

The effects of thyroid hormone deficiency on serum levels and liver messenger RNA (mRNA) expression of insulin-like growth factors (IGFs) were studied in neonatal (until 20 days of life), weaned (22-37 days), and adult (72-87 days) rats, short periods (5, 10, and 15 days) after thyroidectomy (T). Serum levels and liver mRNA expression of IGF-I, plasma and pituitary GH, plasma insulin, and glycemia were measured in all populations; and serum levels and liver mRNA expression of IGF-II were measured only in the neonatal populations. Surprisingly, plasma insulin and GH and serum and liver mRNA expression of IGF-I were found elevated in T neonatal rats, and they decreased in weaned and adult rats and in neonatal rats rendered hypothyroid by mercapto-1-methylimidazole (MMI) treatment (MMI-hypothyroid). T and MMI-treatment of neonatal rats disturbed the normal pattern of progressive decrease of IGF-II with age. A positive correlation between insulin and IGF-I and a poor correlation between GH and IGF-I were found in both hypothyroid neonates (T and MMI-hypothyroid). On the contrary, a positive correlation between GH and IGF-I and a poor correlation between insulin and IGF-I were found for control and T adult rats. Because plasma insulin and GH changed in the same direction in all groups, insulin secretion in T neonatal was suppressed by streptozotocin, and insulin was given to T adult rats. The combined results of these experiments support the idea that the effects of thyroid hormones on IGF-I secretion are age-dependent, and they are mediated mainly by insulin during the neonatal period and by GH during adulthood.