3,5,3'-triiodothyronine (T3) is a survival factor for pancreatic beta-cells undergoing apoptosis

3,5,3'-triiodothyronine (T3) is essential for the growth and the regulation of metabolic functions, moreover, the growth-stimulatory effect of T3 has largely been demonstrated and the pathways via which T3 promotes cell growth have been recently investigated. Type 1 diabetes (T1D) is due to the destruction of beta-cells, which occurs even through apoptosis. Aim of our study was to analyze whether T3 could have an antiapoptotic effect on cultured beta-cells undergoing apoptosis. We have demonstrated that T3 promotes cell proliferation in islet beta-cell lines (rRINm5F and hCM) provoking an increment in cell number (up to 55%: rRINm5F and 45%: hCM), cell viability, and BrdU incorporation, and regulating the cell cycle-related molecules (cyc A, D1, E, and p27(kip1)). T3 inhibited the apoptotic process induced by streptozocin, S-Nitroso-N-Acetylpenicylamine (SNAP), and H2O2 via regulation of the pro- and anti-apoptotic factors Bcl-2, Bcl-XL, Bad, Bax, and Caspase 3. The T3 protective effect was PI-3 K-, but not MAPK- or PKA-mediated, involving pAktThr308. Thus, T3 could be considered a survival factor protecting islet beta-cells from apoptosis.     

3,5,3'-triiodothyronine receptor auxiliary protein (TRAP) enhances receptor binding by interactions within the thyroid hormone response element.

We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half-site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell.     

The effect of adrenaline pretreatment on the in vitro generation of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine (reverse T3) in rat liver preparation

The effects of adrenaline (A) on liver T3 and rT3 neogenesis from T4 were studied in Wistar rats. The animals were implanted subcutaneously either with A or placebo (P) especially coated tablets which linearly released the hormone. The serum A values 6 hrs after implantation of 7.5, 15.0 and 45.0 mg tablets were 6.5 +/- 1.31, 6.8 +/- 1.8 and 16.4 +/- 1.9 ng/ml, respectively vs 4.4 +/- 2.5 ng/ml seen in P pretreated group. The output rates of A were 0.11 (7.5 mg), 0.18 (15 mg) and 0.52 microgram/ml (45 mg). The pretreatment with A led to hyperglycemia and the "low T3 syndrome". Neogenesis of T3 from T4 in medium containing liver microsomes of P pretreated rats was 5.49 +/- 0.25 pmol of T3/mg protein/min and decreased in A pretreated rats to 3.82 +/- 0.17, 3.12 +/- 0.27 and 3.06 +/- 0.11 pmol of T3/mg of protein/min. Neogenesis of rT3 from T4 in microsomes from P group was 1.52 +/- 0.09 pmol rT3/mg protein/min and increased after A to 2.71 +/- 0.11, 2.60 +/- 0.21 and 2.21 +/- 0.34 pmol of rT3/mg protein/min thus showing no dose dependency. Enrichment of microsomes medium with cytosol either from P or A pretreated rats had no effect on T3 generation thus excluding effect of A on cytosolic cofactor. Although cytosol further increased rT3 neogenesis this was seen regardless of whether cytosol was obtained from A or P implanted rats. It is concluded that A decreases the activity of T4-5'-deiodinase in liver, and possibly increases the activity of T4-5-deiodinase.     

Cyclo (His-Pro), a metabolite of thyrotropin-releasing hormone: specific binding to rat liver membranes

[3H]cyclo(His-Pro) bound with high affinity (59 nM) to a single class of sites in rat liver plasma membranes, without significant tracer degradation during equilibration for 60 min at 0 degrees C. Binding was specific and saturable (3.9 pmol/mg protein), and were increased by the addition of K+, Mg++ and Na+ at optimal concentrations, but not of Ca++ at all concentrations tested. In vivo administration of cyclo(His-Pro), but not thyrotropin-releasing hormone, to rats caused the downregulation of cyclo(His-Pro)-binding sites with decreases in specific binding numbers but did not change binding affinity.     

Steroid hormone modulation of 3H-prostaglanding E1 binding to bovine corpus leteum cell membranes.

The specific binding of 3H-prostaglandin E1 (3H-PGE1) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0x10-minus-6M. At concentrations above 2.5 x 10-minus-6M; estrone, 17beta-estradiol (but not 17alpha-estradiol or 17beta-estradiol glucuronide), estroil, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of 3H-PGE1. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced 3H-PGE1 binding. These findings permitted the following correlations between steroid structure and modulation of 3H-PGE1 binding: steroids with a free phenolic ring and a 17beta-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of 3H-PGE1. PGE receptors are heterogeneous with respect to affinity for 3H-PGE1. The steroids that decreased 3H-PGE1 binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased 3H-PGE1 binding caused appearance of new low affinity PGE receptors. Association rate constants for 3H-PGE1 binding were decreased by 17beta-estradiol (61%) and increased by DHT (59%).     

3,5,3'-triiodothyronine (T3) stimulates cell proliferation through the activation of the PI3K/Akt pathway and reactive oxygen species (ROS) production in chick embryo hepatocytes

Thyroid hormones (THs) have a wide variety of essential roles in vertebrates, ranging from the regulation of key metabolic processes to cell proliferation and apoptosis. The classical mechanism of action of THs is genomic; 3,5,3'-triiodothyronine (T3) binds to specific nuclear receptors (TRs) and modifies the expression of specific genes. Recently, a new category of mechanisms, termed nongenomic, has been discovered for T3. These mechanisms include, among others, the rapid activation of signal transduction pathways, such as PI3K/Akt and MAPK, which eventually lead to cell proliferation. These effects are mediated in some cell types by a plasma membrane receptor, identified as integrin αvβ3, and in other cell types by cytoplasmic TRβ1. The aim of this work was to analyze the effect of T3 on the cell growth of chick embryo hepatocytes at two different stages of development, 14 and 19 days, and to determine the activation of the signal transduction pathways, focusing on the potential involvement of a plasma membrane receptor and the possible participation of PI3K/Akt and reactive oxygen species (ROS). Our results clearly show that T3 stimulates cell proliferation at both stages of development through the activation of the PI3K/Akt pathway and the production of small amounts of ROS, which operate as effective second messengers. Moreover, we prove that these effects are not initiated at the plasma membrane receptor for T3.     

Effect of bromocryptine on the secretion of thyrotropic hormone (TSH), prolactin (Pr), human growth hormone (HGH), thyroxine (T4) and triiodothyroxine (T3) in hypothyroidism.

Bromocryptine (CB-154) virtually abolished the rise of serum Pr after TRH stimulation in hypothyroid and euthyroid subjects. The response of serum TSH to TRH stimulation was significantly depressed in hypothyroid but not in euthyroid subjects. No significant changes of serum HGH, T4 and T3 after CB-154 were observed. The dual mode of action of CB-154 in pituitary and hypothalamus is discussed.     

Solubilization of the bombesin receptor from Swiss 3T3 cell membranes. Functional association to a guanine nucleotide regulatory protein

Bombesin and structurally related peptides including gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells. Here we attempted to solubilize bombesin receptors under conditions in which the ligand (125I-labelled GRP) was prebound to the receptor prior to detergent extraction. We found that 125I-GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. These detergents promoted ligand-receptor solubilization in a dose-dependent manner. In contrast, a variety of other detergents including Triton X-100, octylglycoside, CHAPS, digitonin, cholic acid and n-dodecyl-beta-D-maltoside, were much less effective. Addition of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to ligand-receptor complexes isolated by gel filtration enhanced the rate of ligand dissociation in a concentration-dependent and nucleotide-specific manner. Our results demonstrate for the first time the successful solubilization of 125I-GRP-receptor complexes from Swiss 3T3 cell membranes and provide evidence for the physical association between the ligand-receptor complex and a guanine nucleotide binding protein(s).     

Environmental effect on plasma thyroxine (T4), 3,5,3'-triido-L-thyronine (T3), prolactin and cyclic adenosine 3',5'-monophosphate (cAMP) content in the mudskippers Periophthalmus chrysospilos and Boleophthalmus boddaerti

1. In both Periophthalmus chrysospilos and Boleophthalmus boddaerti, T4 was involved in enabling the fish to cope with terrestrial stress and not in osmoregulation in waters of different salinities. In B. boddaerti, however, 3,5,3'-triiodo-L-thyronine (T3) played a more significant role in osmoregulation under the various aquatic conditions. 2. The control of osmoregulation mechanisms in P. chrysospilos kept in waters of different salinities was taken over by prolactin instead, whereas prolactin was only involved in osmoregulation in B. boddaerti under extreme osmotic stress (100% SW). Prolactin is also involved in the terrestrial adaptations of P. chrysospilos. 3. Plasma cAMP levels in P. chrysospilos increased with increasing salinity of the external environment (Tables 4 and 5) implicating its role in the stimulation of chloride secretion and in intracellular isosmotic regulation. 4. Significant increase in the plasma cAMP level of B. boddaerti submerged in 100% SW was also observed. However, the plasma cAMP levels of B. boddaerti fully submerged in 30% and 50% SW were not significantly different from the control as these conditions simulated those of their natural habitats.     

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

Increase in plasma concentrations of 3,5,3'-triiodothyronine and thyroxine after a meal, and its dependence on energy intake

Plasma concentrations of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) were measured before, during and for between 2 and 6 hr following a meal, in young growing piglets. T3 increased after a meal and reached a peak at approximately 60 min. The magnitude of the rise was dependent on both the energy content and nutrient composition of the meal. In animals given either a high or low energy intake baseline values of T3 were similar, whereas there was a difference in the response to a meal (P less than 0.01). Average increases in hormone concentration were 120% (P less than 0.001) and 50% (P less than 0.05) on the high and low intakes respectively. Plasma T4 also increased in those on high intake (P less than 0.025), but no change was detected when the intake was low. The response of T3 to a meal high in either glucose, sucrose, fat or protein was statistically significant except for the protein meal. The rise in T4 after each of these four meals was less consistent, although it did increase significantly after meals high in sucrose or fat. Amongst several possibilities, these results suggest that a meal may induce an increase in secretion of T3 and T4 from the thyroid gland.     

Development of ovarian follicle cells of the stick insect,Carausius morosus br. (Phasmatodea), in relation to their function

The development of follicle cells encompassing the growing oocytes of the stick insect, Carausius morosus (Phasmatodea) has been investigated cytologically and cytophotometrically. The nuclei become 64-ploid through 3 telophasic restitution cycles and 2 endoreduplications. The genomes are evenly distributed over the nucleus, which is also supported by the regular distribution of the nucleoli, and, consequently, fulfill their functions more economically in the (up to 80 μm) thick epithelium. Previtellogenesis is sustained by follicle cells containing 2c-8c DNA, vitellogenesis by 16c cells, vitelline envelope formation by 32c cells, and water uptake, endochorion and exochorion formation by 64c cells.     

Partial characterization of a growth-inhibiting protein in 3T3 cell plasma membranes

A plasma membrane-enriched fraction from 3T3 cells has been detergent-solubilized, and the supernatant of this solubilization was reconstituted into liposomes using soybean lecithin. When these vesicles were added to actively growing cells, cell growth rates were inhibited to levels that were comparable to those observed with the original plasma membranes (at least 50% of maximum growth rates). Liposomes without proteins, or liposomes containing proteins from SV3T3 plasma membranes did not significantly inhibit growth of 3T3 cells. Treatment of the reconstituted vesicles with urea or high concentrations of salt did not eliminate the growth-inhibiting properties of these reconstituted membranes. These results indicate that the specific growth-inhibiting factor in 3T3 cell plasma membranes is a membrane protein that has significant non-polar interactions with the membrane bilayer.     

Specific binding of N-allylnormetazocine (SKF 10047), a ligand of sigma-opioid receptors, with liver membranes

A sigma-opioid receptor ligand, N-allylnormetazocine (SKF 10047), binds specifically and reversibly to rat liver membranes. The rat liver binding sites for SKF 10047 are similar to sigma-opioid CNS receptors. They fail to interact with classical opiates (morphine, naloxone) and opioid peptides but bind with high affinity benzomorphans (bremazocine, SKF 10047) and various psychotropic drugs (haloperidol, imipramine, phencyclidine etc).     

Interleukin-1 binding, internalization, and processing in a murine osteoblastic cell line, MC3T3.E1.

We have investigated the interaction of IL-1 and its receptor on a murine osteoblastic cell line, MC3T3.E1, with regard to binding, internalization, and the fate of the receptor-ligand complex following internalization. Binding experiments indicated that this cell line possesses a high affinity receptor (Kd 1.02 x 10(-10) M) that binds both IL-1 alpha and IL-1 beta, and has approximately 6500 receptors per cell. Cross-linking experiments indicated that the receptor has a molecular weight of 100,000 daltons. Binding of IL-1 to the receptor is inhibited by the Interleukin Receptor Antagonist Protein (IRAP). These characteristics suggest that the murine osteoblastic receptor resembles that found on T lymphocytes and fibroblasts. Internalization experiments showed that this process is fairly rapid and results in degradation of the ligand and subsequent loss of degraded IL-1 from the cell. In this respect, processing of the receptor-ligand complex mimics that observed with IL-1 receptors on murine bone marrow cells, pre-B cells, and macrophages. Although the reasons for these differences are unclear, it may be that, unlike fibroblasts, osteoblasts may function as an effector cell which rapidly removes IL-1 from the immediate environment via ligand degradation while at the same time initiating bone resorption via stimulation of osteopontin biosynthesis.