Kimie Sho

Frameshift mutation, A263fsinsGG, in the hepatocyte nuclear factor-1beta gene associated with diabetes and renal dysfunction.

who had been diagnosed with diabetes at 19 years of age (BMI 18.3 kg/m 2 ) (Fig. LA). This mutation causes a frameshift and the generation of a mutant truncated protein of 264 amino acids that lacks the transcription activation domain. Screening other family members showed that the mutation was also present in the probands sister (IV-3), who had been diagnosed with glycosuria at 14 years of age during the course of an annual school physical examination. The mutation was also found in the probands father (III-2), who had been diagnosed with diabetes at 61 years of age. The two affected sisters (IV-2 and IV-3) are currently being treated with 10-20 U of insulin, and their father with diet. Medical records indicate that the paternal grandmother (D-2) had early-onset diabetes, and so also may have inherited the mutation. The frequency of HNF-1(3 gene mutations in our study population of Japanese subjects who have early-onset type 2 diabetes with a first-degree relative with diabetes is 2.5%. Together with the previous observation by Horikawa et al. (10), our results indicate that mutations in the HNF-ip gene are not a major cause of early-onset type 2 diabetes in Japanese, although the results apply only to the coding region of the HNF-1(3 gene. The proband and her sister had decreased renal function (creatinine clearance <50 ml/min) and multiple bilateral renal cysts with three to five cysts per kidney (0.3-1.2 cm in diameter) (Fig. IE). Their father also had chronic renal failure with renal cysts, which is now being treated by hemodialysis. While the two affected sisters showed no evidence of proteinuria, the father had persistent mild proteinuria (<300 mg/day), which was first noticed during the course of an annual health examination 20 years before the diagnosis of diabetes. The A263fsinsGG mutation was also found in the nondiabetic 2-year-old son (V-l) of the proband and the 5-year-old daughter (V-2) of the affected sister, who also had elevated levels of serum creatinine (1.3-1.8 mg/dl; normal 0.6-1.0 mg/dl) at birth. Moreover, subject V-l was found at birth to have bilateral renal cysts measuring several millimeters in diameter, and fetal ultrasound examination at 27 weeks of gestation revealed renal abnormalities, including bilateral enlarged kidney mass and multiple parenchymal hyperechoic nodules (Fig. LET). These observations suggest that the A263fsinsGG mutation in the HNF-ip gene might be associated with diabetes and renal dysfunction in this family. Renal dysfunction described in this report is defined broadly, including proteinuria, decreased renal function, and renal cysts. Moreover, these abnormalities appear to precede the onset of dia

Sphingosine 1-Phosphate Stimulates Hydrogen Peroxide Generation through Activation of Phospholipase C-Ca2+ System in FRTL-5 Thyroid Cells: Possible Involvement of Guanosine Triphosphate-Binding Proteins in the Lipid Signaling

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(l-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), but not in its absence. Guanosine 5′-O-(2-thiodiphosphate) inhibited the S1P-induced GTPγS-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-...

Stimulation of adenosine receptor enhances α1 -adrenergic receptor-mediated activation of phospholipase C and Ca2+ mobilization in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells

Norepinephrine (NE) stimulated FRTL‐5 thyroid cells via an α1‐adrenergic receptor, resulting in cytosolic Ca2+ ([Ca2+]i) mobilization and activation of phospholipase C. Adenosine and its receptor agonist, phenylisopropyladenosine (PIA), although not exerting a direct effect, markedly enhanced the NE‐induced changes. Basal NE action was not totally abolished whereas the permissive action of adenosine and PIA was completely abolished by pretreatment of the cells with islet‐activating protein (IAP), pertussis toxin. The decrease in cAMP level induced by adenosine or PIA is not the cause of their permissive effect, since this effect was not reversed by the addition of cAMP‐increasing agents. We conclude that an IAP substrate GTP‐binding protein(s) plays a novel role in forming a stimulatory coupling between an adenosine receptor and an α1‐adrenergic receptor‐coupled phospholipase C system.

Reciprocal modulation of thyrotropin actions by P1-purinergic agonists in FRTL-5 thyroid cells. Inhibition of cAMP pathway and stimulation of phospholipase C-Ca2+ pathway.

In FRTL-5 thyroid cells, thyrotropin (TSH) stimulates I- efflux in association with phospholipase C activation and Ca2+ mobilization. TSH also stimulates DNA synthesis, accompanied by cAMP accumulation. Significant activation of the phospholipase C-Ca2+ pathway requires 10-100 nM TSH a concentration 10(3) to 10(4) times higher than necessary to stimulate the cAMP pathway. When the P1-purinergic agonist, phenylisopropyladenosine (PIA) is added to the reaction medium, the former pathway is markedly enhanced, whereas the latter pathway is inhibited. As a result, in the presence of PIA, both TSH-induced pathways are activated at similar TSH concentrations. These PIA actions are completely reversed by a prior treatment of cells with islet-activating protein (IAP); pertussis toxin. When adenosine deaminase is added to the reaction medium, TSH-induced cAMP accumulation is significantly enhanced, suggesting an autocrine action of adenosine. In IAP-treated cells, the level of TSH-induced cAMP accumulation reaches that of deaminase-treated control cells, and no further increase is observed when adenosine deaminase is added. We conclude that in the thyroid, either an neural or autocrine adenosine signal, mediated by an IAP-sensitive G-protein, switches TSH signal transduction from the cAMP pathway to the phospholipase C-Ca2+ pathway.

Involvement of pertussis toxin‐sensitive GTP‐binding proteins in sphingosine 1‐phosphate‐induced activation of phospholipase CCa2+ system in HL60 leukemia cells

Exogenous sphingosine 1‐phosphate (S1P) induced Ca2+ mobilization, in association with an increase in inositol polyphosphate production reflecting activation of phospholipase C in HL60 leukemia cells. The increase in intracellular Ca2+ concentration ([Ca2+]i) induced by S1P was inhibited by an appropriate treatment of the cells with pertussis toxin (PTX), U73122 (a phospholipase C inhibitor) or phorbol 12‐myristate 13‐acetate (PMA). In parallel with the Ca2+ response, these agents also inhibited inositol polyphosphate production. The S1P‐induced Ca2+ response was also attenuated in the dibutyryl cAMP‐induced differentiated cells, where GTP‐binding protein‐induced Ca2+ response is suggested to be enhanced. Lysophosphatidic acid (LPA) also increased [Ca2+]i in the cells, but the maximal response was about half of that of S1P, and furthermore PTX and dibutyryl cAMP treatment hardly affected the LPA‐induced Ca2+ mobilization. We conclude that exogenous S1P mobilizes Ca2+ through phospholipase C activation. The S1P‐induced enzyme activation is at least partly mediated by PTX‐sensitive GTP‐binding protein‐coupled receptors which may be different from LPA receptors.

Loss-of-function and Dominant-negative Mechanisms Associated with Hepatocyte Nuclear Factor-1β Mutations in Familial Type 2 Diabetes Mellitus

Hepatocyte nuclear factor (HNF)-1β, a homeodomain-containing transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues. Recent genetic studies have identified two HNF-1β mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes. Despite the defects being in the same gene, diverse severities of disease are observed in the affected subjects. To investigate the molecular mechanism by which mutations might cause various phenotypic features, wild type and mutant proteins were transiently expressed in insulin-producing (MIN6) and hepatic (HepG2) cells. Luciferase reporter assay showed that both mutations resulted in a marked reduction of transactivation activity. Because their dimerization activity was found to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with wild type, both of the mutants significantly decreased wild type activity in HepG2 cells. In contrast, although A263fsinsGG functioned similarly in MIN6 cells, R177X failed to affect wild type activity in this cell line. Immunohistochemical analysis of the mutants suggests that this functional divergence might be generated by the modification of nuclear localization. These results suggest that HNF-1β mutations may impair pancreatic β-cell function by loss-of-function and dominant-negative mechanisms.

Insulin modulates thyrotropin-induced follicle reconstruction and iodine metabolism in hog thyroid cells cultured in a chemically defined medium

Effects of insulin on cultured hog thyroid cells were examined in a chemically defined medium supplemented with thyrotropin. Thyrotropin-induced follicle reconstruction and recovery of functional activities were stimulated by the addition of insulin to the culture medium. The iodide uptake and organification at the maximum stimulation exceeded 3 times those of the control values. The minimum effective dose of insulin was 60 microU/ml. The insulin stimulation occurred after a latency period of 24 h. Since no growth promoting effect of insulin was observed during the cultivation period in the present study, insulin appears to act in concert with thyrotropin in regenerating the characteristic properties of the differentiated cells during cultivation.

An adenosine receptor agonist-induced modulation of TSH-dependent cell growth in FRTL-5 thyroid cells mediated by inhibitory G protein, Gi

Adenosine has been shown to modulate the TSH-induced DNA synthesis in FRTL-5 thyroid cells. The mechanism of this adenosine action has been somewhat controversial because both A1 adenosine receptor-mediated and non-receptor-mediated mechanisms have been proposed. We have now reexamined our preliminary finding of the inhibitory action of a non-metabolizable adenosine derivative, N6-(L-2-phenylisopropyl)adenosine (PIA), on the TSH-induced DNA synthesis to clarify the adenosine-dependent mechanism of cell growth modulation. PIA dose-dependently inhibited the TSH-induced DNA synthesis expressed by [3H]thymidine incorporation into DNA. This adenosine derivative also prevented the TSH-induced entry of the cell cycle to the S phase at 24 h of culture and the increase in cell number at 48 h. These PIA actions on different aspects of TSH-dependent cell growth were abolished by the treatment of the cells with pertussis toxin, suggesting the involvement of Gi in the PIA action mechanism. Dibutyryl cAMP-induced DNA synthesis was not influenced by PIA. In concert with our previous finding that PIA in a similar concentration range inhibited TSH-induced cAMP production through the adenosine A1 receptor, the present results strongly support the idea that the major pathway of adenosine signaling for the inhibition of the TSH-induced cell proliferation is through the A1 adenosine receptor-Gi system.

Evidence for a preferential iodination site within the thyroglobulin molecule

Mouse 330 kDa thyroglobulin labeled in vivo was analyzed using a tryptic peptide mapping technique and high performance liquid chromatography (HPLC). 30 min after Na125I injection, one peptide spot (spot 7) on a silica gel plate was the only prominent labeled peptide, followed by other labeled peptide spots after 1 h. HPLC showed that spot 7 was rich in monoiodotyrosine. The ratios between the iodoamino acids were strictly maintained from 1 to 6 h after Na125I injection. Spot 7 was again the first spot that appeared from the samples of iodine-deficient mice. These data indicate that there is some preferential iodination site(s) within the thyroglobulin molecule and also that their iodoamino acid composition is predetermined.

P1-Purinergic Receptor-Mediated Modulation of TSH Actions on FRTL-5 Thyroid Cells: Possible Switching From cAMP Pathway to Inositol Phosphate-Ca System

Abstract In thyroid cells, a PI-agonist, via G1 like protein, enhanced a TSH-induced I−-efflux by intensifying a TSH-dependent inositol polyphosphate production followed by a Ca2+ mobilization, but diminished a TSH-dependent DNA synthesis by attenuating a TSH-dependent cAMP accumulation.