Thyromimetic actions of tetrabromobisphenol A (TBBPA) in steatotic FaO rat hepatoma cells.

Tetrabromobisphenol A (2,2-bis(3,5-dibromo-4-hydroxyphenyl propane-TBBPA) is the most produced brominated flame retardant, detected in the environment and in biological samples. TBBPA shares structural similarities with thyroid hormones (THs), and it has been shown to interfere with different aspects of TH physiology, this raising concern on its possible effects as an endocrine disruptor in humans and wildlife. THs play a major role in lipid metabolism, with the liver representing one of their main target tissues. At the cellular level, THs act through interactions with TH receptors (TRs), as well as through TR-independent mechanisms. Rat hepatoma FaO cells (a liver cell line defective for functional TRs) overloaded with lipids have been utilized as a model to investigate the anti-steatotic effects of THs in the hepatocyte. In this work, the possible effects of TBBPA in steatotic FaO cells were investigated. Exposure to TBBPA for 24 h reduced triglyceride (TAG) content and the size of lipid droplets (LDs); similar effects were obtained with equimolar doses (10(-6) M) of T3 (3,3',5-L-triiodothyronine). TBBPA and T3 showed common effects on transcription of genes involved in lipid homeostasis. In particular, TBBPA mainly up-regulated mRNA levels for LD-associated oxidative tissue-enriched PAT protein (OXPAT), peroxisome proliferator-activated receptor (PPAR) isoform ß/δ, and the mitochondrial uncoupling protein 2 (UCP2). The results demonstrate that TBBPA can decrease lipid accumulation in steatotic cells through stimulation of oxidative pathways. These data identify novel thyromimetic actions of TBBPA at the cellular level.

Laser diode array pumped continuous wave Rubidium vapor laser.

We have demonstrated continuous wave operation of a laser diode array pumped Rb laser with an output power of 8 Watts. A slope efficiency of 60% and a total optical efficiency of 45% were obtained with a pump power of 18 Watts. This laser can be scaled to higher powers by using multiple laser diode arrays or stacks of arrays.

3,5-diiodothyronine mimics the effect of triiodothyronine on insulin-like growth factor binding protein-4 expression in cultured rat hepatocytes.

We have previously demonstrated that triiodothyronine (T(3)) stimulates hepatic IGFBP-4 expression in rats. Since there is evidence that some of the genes whose expression is regulated by T(3) are also sensitive to 3,5-diiodothyronine (T(2)), we used the adult rat hepatocyte model in primary cultures directly exposed to T(2) to evaluate insulin-like growth factor binding protein-4 (IGFBP-4) expression by Northern and Ligand blot analyses in this study. Our results demonstrate that T(2), like T(3), is able to enhance IGFBP-4 mRNA and protein after 12-24 h of incubation. The potency of the two iodothyronines is comparable as judged by dose-dependence experiments. The T(2)-induced IGFBP-4 increase is independent from ongoing protein synthesis but dependent on active transcription. Since T(3) and T(2) do not affect IGF-I production, it appears that the iodothyronines affect the hepatic IGF system at the IGFBP level. Our data, demonstrating that T(2) mimics the stimulatory effect of T(3) on IGFBP-4 expression by rat hepatocytes, allow us to include IGFBP-4 gene among the genes regulated by the two iodothyronines.

Retinoic acid increases insulin-like growth factor-binding protein-4 expression in cultured rat hepatocytes.

Hepatic insulin-like growth factor binding protein (IGFBP) expression is controlled by diverse factors including thyroid hormone, which enhances IGFBP-4 production in hepatocytes. In the present work, we have investigated whether hepatic IGFBP-4 expression is regulated by retinoic acid (RA), which acts via nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily. Primary cultures of adult rat hepatocytes were incubated with two natural stereoisomers of RA, all-trans RA and 9-cis RA (atRA and 9cRA), and with the synthetic RA receptor (RAR)-selective agonist TTNPB. IGFBP-4 mRNA abundance was measured by Northern blot and protein production was evaluated by Ligand blot on hepatocyte-conditioned culture media. Our results indicate that atRA, 9cRA, and TTNPB increase IGFBP-4 expression by cultured hepatocytes, both at the mRNA and protein level. The RARs play a definite role in this regulation, which is independent from ongoing protein synthesis but dependent on active transcription. AtRA and thyroid hormone act synergistically in increasing hepatic IGFBP-4 expression. Our data establish a role for hormonal factors such as thyronines and retinoids in regulating the hepatic IGF system directly at the IGFBP-4 level.

Uncoupling protein 2 mRNA expression and respiratory parameters in Kupffer cells isolated from euthyroid and hyperthyroid rat livers.

OBJECTIVE: The levels of uncoupling protein 2 (UCP2) mRNA and determinants of respiration (ATP synthesis, proton leak and non-mitochondrial respiration) were evaluated in Kupffer cells isolated from the livers of normal euthyroid, acute hyperthyroid and chronic hyperthyroid rats. METHODS: After liver perfusion, Kupffer cells were purified by density-gradient centrifugation followed by counterflow centrifugal elutriation. UCP2 mRNA levels were measured by Northern blot and respiratory parameters by polarographic method. RESULTS: In cells isolated from hyperthyroid (tri-iodothyronine (T(3))-treated) rats, the effect of T(3) treatment on the UCP2 mRNA level varied: it was more than doubled (P<0.05) in acutely T(3)-treated rats but, after chronic (3-week) T(3) treatment, it was only 30% (not statistically significant) above the control (euthyroid) level. In Kupffer cells from the livers of chronic hyperthyroid rats, we observed an increase in total respiration rate, with an increase in the percentage attributable to the proton leak and a corresponding decrease in the percentage attributable to ATP synthesis (no alteration was observed in the percentage attributable to non-mitochondrial respiration). In the acute hyperthyroid rats, no significant differences were observed in any of the respiratory parameters, although they all tended to increase. CONCLUSION: These data are indicative of a possible uncoupling effect of UCP2 in Kupffer cells. T(3), by enhancing the expression of UCP2, could play a role in the energy homeostasis of these cells.

Regulation of renal growth and IGFBP-4 expression by triiodothyronine during rat development.

The insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs), which regulate IGF activity, play a fundamental role in renal cell proliferation and differentiation. The thyroid hormone is considered to be required for kidney development; excess induces local hypertrophy and hyperplasia. The aim of the present study was to investigate the possible involvement of the IGF/IGFBP system in thyroid hormone-induced renal growth during the development of the rat. Our results show that thyroid hormone withdrawal by 6-propyl-2-thiouracil (PTU)-treatment of rats at all ages had no effect on renal IGFBP-4 mRNA levels, whereas the abundance of the serum protein was decreased compared to controls. Intraperitoneal triiodothyronine (T3) administration to hypothyroid rats resulted in renal hypertrophy associated with a significant upregulation of IGFBP-4 expression with increased levels of renal IGFBP-4 mRNA and serum protein. T3-induced upregulation of IGFBP-4 expression suggests the involvement of the local IGF/IGFBP system in T3-induced renal hypertrophy.

Increased insulin-like growth factor binding protein-4 expression after partial hepatectomy in the rat.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important regulators of cell growth produced by different tissues. The IGFBPs regulate cell growth by modulating the activity and bioavailability of IGFs. The evidence that IGFBP-1 is a liver-specific immediate-early gene highly induced after 70% partial hepatectomy (PHx) suggests a role for the IGF-IGFBP system in hepatic regeneration. In this work we analyzed the effect of PHx on the expression of IGFBP-4, which is highly produced by the liver and very abundant in rat serum. Our results show a marked increase in hepatic IGFBP-4 mRNA levels 6-12 h after PHx and no significant change in sham-operated control animals. A parallel rise in IGFBP-4 transcript abundance was observed in the kidneys of PHx rats but not in sham-operated animals. Moreover, ligand blot analysis demonstrated that serum IGFBP-4 levels began to increase 12-24 h after surgery, consistent with the rise in the corresponding mRNA. This enhancement in IGFBP-4 production after PHx could be part of a fine regulatory mechanism to modulate IGF activity during liver regeneration.

IGF-I production by adult rat hepatocytes is stimulated by transforming growth factor-alpha and transforming growth factor-beta1.

Previously, we have observed that epidermal growth factor (EGF), a potent mitogen for cultured hepatocytes, stimulates the production of IGF-I and IGF-binding proteins (IGFBPs) by cultured hepatocytes from adult rats. This study was undertaken to investigate the possibility that other growth factors of hepatic origin could specifically be involved in the regulation of IGF-I and IGFBP expression. The effects of transforming growth factor-alpha (TGF-alpha), through EGF receptors to induce a mitogenic response, and transforming growth factor-beta1 (TGF-beta1), produced by non-parenchymal liver cells and able to inhibit hepatocyte proliferation in vivo and in culture, have been studied in cultured adult rat hepatocytes. Our results demonstrate that TGF-alpha and TGF-beta1 significantly stimulate IGF-I and IGFBP secretion by cultured hepatocytes but no change in the abundance of IGF-I and IGFBP mRNAs was observed with respect to controls. Cycloheximide is able to inhibit both basal and TGF-stimulated release of IGF-I and a similar effect was elicited by octreotide, the somatostatin analog, known to directly affect hepatic IGF-I gene expression. Our findings show the role of the liver in the secretion of IGF-I and IGFBPs, not only under endocrine and nutritional control but also under autocrine and paracrine control.

Temporary clipping in the surgery of endocranial aneurysms.

The authors' experience in the routine use of temporary clipping procedures in the surgery of endocranial aneurysms is reported. To analyse the validity of such a method we compared the outcome in a series of 153 aneurysms operated according to the traditional procedure (temporary clipping of the afferent vessel only in the case of intraoperative rupture of the aneurysmatic sac) with that of a more recent series of 225 in which the procedure was applied routinely. An unsatisfactory surgical outcome was found in 12.5% and 6.6% of patients respectively, with a corresponding unfavourable outcome in 5.6% and 2.6%.

Effect of the somatostatin analog, octreotide, and of other hormones on the release of the acid-labile subunit of the 150 kDa complex by rat hepatocyte in primary culture.

OBJECTIVE: In normal subjects, the major form of circulating IGF is the GH-dependent 150 kDa complex. The liver appears to be the main source of the three components of the 150 kDa complex and, in particular, hepatocytes synthesize the insulin-like growth factor (IGF) peptide and the acid-labile subunit (ALS), whereas Kupffer and sinusoidal endothelial cells produce IGF-binding protein-3 (IGFBG-3). We have studied the effects of the somatostatin analog octreotide, IGF-II des(1-3)IGF-I, transforming growth factor (TGF)-beta 1 and tri-iodothyronine (T3) on ALS secretion into the medium conditioned by rat hepatocytes in primary culture. METHODS: The regulation of ALS release was evaluated in the conditioned medium of adult rat hepatocytes exposed to increasing concentrations of test substances or to vehicle alone (control), after gel filtration in basic conditions, by immunoblot using an antiserum generated against the N-terminal 34 amino acids of human ALS. RESULTS: The results demonstrate that: 1) octreotide in vitro produces a dose-dependent inhibition of both basal and GH-stimulated ALS secretion into the hepatocyte conditioned medium; 2) the release of ALS by adult rat hepatocytes is not affected by the presence during the incubation of des(1-3)IGF-I or IGF-II; 3) an inhibitory effect, although only with very high doses, can be observed after treatment with TGF-beta 1; and 4) a small but significant increase of ALS released into the medium can be seen when hepatocytes are treated with T3. CONCLUSIONS: Evaluation of the effect of substances known to affect the production of IGF peptides, the IGFBPs, or both, on adult rat hepatocytes in primary culture revealed no powerful stimulator, but instead a potent inhibitor of ALS release/synthesis. Our data suggest that the effect of somatostatin on the 150 kDa complex is mediated not only by the reduction in GH concentration, but also by a direct inhibition of ALS release or synthesis.

Tri-iodothyronine increases insulin-like growth factor binding protein-4 expression in rat hepatocytes.

Previous in vivo studies demonstrated significant variations in insulin-like growth factor binding protein-1 (IGFBP-1), IGFBP-2 and IGFBP-4 hepatic mRNAs and/or serum levels depending on the rat thyroid status. In this study we employed cultured hepatocytes from adult rats to demonstrate a possible direct regulation of these genes by tri-iodothyronine (T3). Northern blot analysis revealed that IGFBP-1 and -4 messages were clearly expressed, whereas IGFBP-2 signal was barely detectable. No significant effects on IGFBP-1 mRNA level or on peptide secretion were detected in T3-cultured hepatocytes. In contrast, significant increases in IGFBP-4 mRNA steady-state levels as well as in IGFBP-4 secretion were observed in hepatocytes cultured for 12-24 h in the presence of T3. The T3 effect on IGFBP-4 transcript levels appears to consist of enhanced gene transcription and is independent of ongoing protein synthesis. The T3-increased IGFBP-4 expression in cultured hepatocytes is consistent with our in vivo experiments demonstrating an increase in hepatic IGFBP-4 mRNA and serum IGFBP-4 levels in T3-treated rats. Furthermore, significant decreases in hepatic IGFBP-4 message and serum IGFBP-4 levels were observed in hypothyroid rats compared with euthyroid controls. Our data establish an important direct role for thyroid hormone in regulating IGFBP-4 expression and consequently IGF activity.

Regulation of IGFBP-1 and -4 expression by triiodothyronine (T3) in cultured hepatocytes is cell- and gene-specific.

Evidence suggests that thyroid hormone plays a role in the regulation of hepatic IGF/IGFBP expression both in human and rats. In this study we compared the effect of T3 on IGFBP-1 and -4 expression in rat hepatocyte primary cultures and in the human hepatoma cell line HepG2. Northern blot analysis revealed that IGFBP-1 mRNA levels were not affected by T3 in cultured rat hepatocytes, whereas a net increase of IGFBP-1 transcript abundance was induced by the hormone in HepG2 cells. On the contrary, IGFBP-4 mRNA levels were increased in rat hepatocytes cultured in the presence of T3, but unaffected in T3-treated HepG2 cells. Therefore, thyroid hormone seems to regulate hepatic IGFBP expression in a direct and gene-specific way. Moreover, the effects of thyroid hormone depend strictly on the source of target hepatocyte.

Relationship between DNA synthesis and growth factor expression in primary cultures of adult rat hepatocytes.

In this study we employed primary culture of adult rat hepatocytes to verify the effects of two different extracellular matrices (collagen, matrigel) on EGF-stimulated DNA synthesis and c-myc expression. Our results confirm that in adult rat hepatocytes EGF induces DNA synthesis, preceded by a transient increase of c-myc expression, when cells are cultured at low density on collagen. DNA synthesis appears to be in reciprocal relationship with hepatic expression of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-4, suggesting that IGF-I/IGFBPs system is not involved in liver growth.

Functions and regulation of the acid-labile subunit of the 150 K complex.

In normal subjects, the major form of circulating IGF is the GH-dependent 150 K complex. As demonstrated by gel-permeation chromatography, the acid-labile subunit (ALS) purified from human serum, incubated for 2 h at 20 degrees C with [125I]IGF-I and rIGFBP-3, is able to increase not only the molecular weight (mol. wt.) of the IGF-IGFBP-3 complex, but also the amount of IGF-I bound. In both charcoal and polyethylene glycol ligand binding assays, competitive binding curves for the displacement of [125I]IGF-I from rIGFBP-3 by increasing concentrations of unlabeled IGF-I showed an increased binding activity of rIGFBP-3 in the presence of ALS. The effect of ALS on rIGFBP-3 binding activity was dose dependent. In addition, ligand and immunoblot revealed that ALS and rIGFBP-3 are able to form a high mol. wt. complex in the absence of IGF peptide. On the basis of these data, ALS seems to have a more complex function than that of simply increasing the mol. wt of the IGF-IGFBP-3 complex. The regulation of ALS synthesis by rat hepatocytes in primary culture has also been evaluated by immunoblot. In agreement with the in vivo finding of an inhibitory effect of octreotide (a somatostatin analog) on the formation of the 150 K complex in acromegalic subjects, we could observe in vitro that octreotide produces a dose-dependent inhibition of ALS secretion into the hepatocyte conditioned medium. TGF-beta 1 was also inhibitory at high doses. On the contrary, we could not evidence any effect of IGF-I or IGF-II, while a small increase has been noted after incubation with T3.

Thyroid hormone modulates the expression of rat liver gammaglutamyltranspeptidase activity.

Gammaglutamyltranspeptidase (GGT) activity is considered as a marker of liver phenotype, being maximally expressed in fetal liver and virtually absent in adult mature tissue. Since thyroid hormone has been identified to influence growth and development of almost all tissues, we have investigated the possible involvement of such factors in regulating rat hepatic GGT. Our results indicate that the activity of GGT in liver of perinatal hypothyroid rats is low as well as in control animals; instead, it is greatly increased in adult hypothyroid rats compared to controls of the same age (+260% in 2-month-old hypothyroid rats). Replacement therapy with T3 to hypothyroid rats normalizes the enzyme activity to the levels of control animals. Thyroid hormone appears to modulate the gene expression of the enzyme, since in the different thyroid status GGT mRNA level closely parallels the variations of the enzyme activity. These results further suggest that thyroid hormone plays a crucial role in maintaining the phenotype of adult liver tissue.

Role of growth factors in the human endometrium during aging.

The aim of this study was to investigate the possible role of epidermal growth factor (EGF) and of insulin-like growth factor-I (IGF-I) in physiological and pathological changes of the endometrial tissue during aging. Thirty-four patients undergoing hysterectomy were divided into three groups: (A) premenopausal women with regular menses, (B) pre-menopausal women with irregular bleeding and (C) post-menopausal women. Endometrial samples were collected after the removal of uterus and were used for immunohistochemical evaluation of EGF, EGF receptor (EGFr) and IGF-I and also for Northern blot analysis of IGF-I gene expression. Plasma levels of 17 beta-oestradiol (E2), D4-androstenedione (D4-A) and oestrone (E1) were also assayed. The immunohistochemical scores (HSCORES) for EGF, EGFr and IGF-I were significantly higher in groups A and B than in group C. Independently from the menstrual history, significantly higher HSCORES of EGF, EGFr and IGF-I were present in hyperplastic endometrium than in those which were proliferative and atrophic. Moreover, IGF-I mRNA expression was observed in all pre-menopausal women, whereas only 1 post-menopausal women with hyperplastic endometrium showed detectable RNA encoding for IGF-I. Higher levels of D4-A were also significantly correlated (P < 0.05) with higher HSCORES of EGF, EGFr and IGF-I. Our results suggest that the above mentioned growth factors could act as mediators of oestrogens on the endometrial functional activity.

Effect of epidermal growth factor on insulin-like growth factor-I (IGF-I) and IGF-binding protein synthesis by adult rat hepatocytes.

Growth hormone has been established as a primary regulator of IGF-I gene expression in adults, not only in liver but also in many extrahepatic tissues. We considered the possibility that IGF-I production by adult rat liver could also be stimulated by epidermal growth factor (EGF), a peptide known to be involved in liver regeneration. Chromatographic analysis performed after acid treatment of conditioned media revealed the presence of both immunoreactive (IR) IGF-I and IGF binding protein (IGFBP). Both IR IGF-I and IGFBP were present in the conditioned medium of adult rat hepatocytes in basal conditions. The stimulation of IGF-I and IGFBP secretion by EGF appears to be dose-dependent with a significant increment already evident at 5 nM. That EGF stimulates secretion is supported by the finding that IGF-I and IGFBP-1 mRNA levels are increased after EGF supplementation. We conclude that adult rat hepatocytes spontaneously produce IGF-I and IGFBP, and that EGF is able to increase their synthesis and secretion. This non-growth hormone-dependent regulation of IGF-I and IGFBP-1 production by adult rat hepatocytes in culture indicates an important autocrine/paracrine role for IGF-I, particularly during liver regeneration after extensive organ mass loss.