II - Insulin processing in mitochondria.

Our objective was to know how insulin is processing in mitochondria; if IDE is the only participant in mitochondrial insulin degradation and the role of insulin degradation on IDE accumulation in mitoplasts. Mitochondria and its fractions were isolated as described by Greenwalt. IDE was purified and detected in immunoblot with specific antibodies. High insulin degradation was obtained through addition to rat's diet of 25 g/rat of apple and 10 g/rat of hard-boiled eggs, 3 days a week. Mitochondrial insulin degradation was assayed with 5 % TCA, insulin antibody or Sephadex G50 chromatography. Degradation was also assayed 60 min at 37 °C in mitochondrial fractions (IMS and Mx) with diet or not and without IDE. Degradation in fractions precipitated with ammonium sulfates (60-80 %) were studied after mitochondrial insulin incubation (1 ng. insulin during 15 min, at 30 °C) or with addition of 2.5 mM ATP. Supplementary diet increased insulin degradation. High insulin did not increase mitoplasts accumulation and did not decrease mitochondrial degradation. High insulin and inhibition of degradation evidence insulin competition for a putative transport system. Mitochondrial incubation with insulin increased IDE in matrix as observed in immunoblot. ATP decreased degradation in Mx and increased it in IMS. Chromatography of IMS demonstrated an ATP-dependent protease that degraded insulin, similar to described by Sitte et al. Mitochondria participate in insulin degradation and the diet increased it. High insulin did not accomplish mitochondrial decrease of degradation or its accumulation in mitoplasts. Mitochondrial incubation with insulin increased IDE in matrix. ATP suggested being a regulator of mitochondrial insulin degradation.

I - insulin transfer to mitochondria.

The aim of this study was to determine if insulin is transferred to mitoplasts by insulin-degrading enzyme (IDE).Hepatic mitochondria were isolated and controlled by electron microscopy. IDE was obtained from rats muscle by successive chromatography steps. Insulin accumulation in mitoplasts and outer membrane + intermembrane space (OM + IMS) was studied with (125)I-insulin. Mitochondrial insulin accumulation and degradation was assayed with Sephadex G50 chromatography, insulin antibody and 5 % TCA. Mitoplasts and OM + IMS were isolated with digitonin. Insulin accumulation was studied at 25 °C at different times, without or with IDE, Bacitracin, 2,4-dinitrophenol, apyrase or sodium succinate + adenosine diphosphate. Insulin accumulation in mitoplasts and OM + IMS after mitochondrial cross-linking was studied with electrophoresis in SDS-PAGE, immunoblots of IDE, insulin or TIM23 (inner mitochondrial transporter) and autoradiography.The studies showed that addition of IDE increased insulin transfer from OM + IMS to mitoplasts, and the insulin accumulation in mitoplast was IDE dependent. Bacitracin and 2,4-dinitrophenol decreased this transfer. The [Insulin-IDE] complex and [Mitoplasts] was studied as a bimolecular reaction following a second order reaction. The constant "k" (liter.mol⁻¹ s⁻¹) showed that IDE increased and Bacitracin or 2,4-dinitrophenol decreased the velocity of insulin transfer. SDS-PAGE and immunoblots studies showed bands and radioactivity coincident with IDE, insulin and TIM23. Non degraded insulin was demonstrated in immunoblot after IDE immunoprecipitation from mitoplasts. Confocal studies showed mitochondrial colocalization of IDE and insulin.The results showed that insulin at 25 °C were transferred from OM + IMS to mitoplasts by IDE or that the enzyme facilitates this transfer, and they reach the matrix together.

Changes in hepatic lipogenic and oxidative enzymes and glucose homeostasis induced by an acetyl-L-carnitine and nicotinamide treatment in dyslipidaemic insulin-resistant rats.

Normal rats fed a sucrose-rich diet (SRD) develop dyslipidaemia and insulin resistance. The present study examined whether administration of the mitochondrial nutrients nicotinamide and acetyl-L-carnitine reversed or improved these metabolic abnormalities. Male Wistar rats were fed an SRD for 90 days. Half the rats then received daily injections of nicotinamide (25 mg/kg, i.p.) and acetyl-L-carnitine (50 mg/kg, i.p.) for a further 90 days. The remaining rats in the SRD-fed group and those in a normal chow-fed control group were injected with an equal volume of saline solution for the same period. The following parameters were determined in all groups: (i) liver activity of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and carnitine-palmitoyl transferase-1 (CPT-1); (ii) hepatic and skeletal muscle triacylglycerol content, plasma glucose, insulin, free fatty acid (FFA) and triacylglycerol levels and pancreatic insulin content; and (iii) glucose tolerance. Administration of nicotinamide and acetyl-L-carnitine to the SRD-fed rats reduced dyslipidaemia, liver steatosis, muscle triacylglycerol content and hepatic FAS and ACC activities and increased CPT-1 activity. In addition nicotinamide and acetyl-L-carnitine improved the glucose disappearance rate (K(g)), normalized plasma glucose levels and moderately increased insulinaemia without altering pancreatic insulin content. Finally, nicotinamide and acetyl-l-carnitine administration reduced bodyweight gain and visceral adiposity. The results of the present study suggest that altering key hepatic lipogenic and fatty acid oxidative enzymatic activity could improve dyslipidaemia, liver steatosis and visceral adiposity. Indeed, administration of nicotinamide and acetyl-l-carnitine improved glucose intolerance and normalized plasma glucose levels.

Effect of dexamethasone and testosterone treatment on the regulation of insulin-degrading enzyme and cellular changes in ventral rat prostate after castration.

Insulin-degrading enzyme (IDE) has been shown to enhance the binding of androgen and glucocorticoid receptors to DNA in the nuclear compartment. Glucocorticoids cause hyperglycaemia, peripheral resistance to insulin and compensatory hyperinsulinaemia. The aim of the present study was to investigate the effect of dexamethasone (D), testosterone (T) and dexamethasone plus testosterone (D + T) on the regulation of IDE and on the remodelling of rat ventral prostate after castration (C). Castration led to a marked reduction in prostate weight (PW). Body weight was significantly decreased in the castrated animals treated with dexamethasone, and the relative PW was 2.6-fold (±0.2) higher in the D group, 2.8-fold (±0.3) higher in the T group and 6.6-fold (±0.6) higher in the D + T group in comparison with the castrated rats. Ultrastructural alterations in the ventral prostate in response to androgen deprivation were restored after testosterone and dexamethasone plus testosterone treatments and partially restored with dexamethasone alone. The nuclear IDE protein level indicated a 4.3-fold (±0.4) increase in castrated rats treated with D + T when compared with castration alone. Whole-cell IDE protein levels increased approximately 1.5-fold (±0.1), 1.5-fold (±0.1) and 2.9-fold (±0.2) in the D, T and D + T groups, respectively, when compared with castration alone. In conclusion, the present study reports that dexamethasone-induced hyperinsulinaemic condition plus exogenous testosterone treatment leads to synergistic effects of insulin and testosterone in the prostatic growth and in the amount of IDE in the nucleus and whole epithelial cell.

Un enfoque para un problema sanitario y social: yodurias en embarazadas de una región yododeficiente / An approach to a sanitary and social problem: urinary iodine excretion in pregnant women from a iodine deficient region

La yoduria o eliminación urinaria de yodo (EUI) es un método efectivo para detectar el déficit en su ingesta. En 121 embarazadas se midió yoduria en dos muestras (matutina y vespertina) con el método turbidimétrico modificado por Pino. Se consideró normal una EUI =150 ug/l. En las mujeres con EUI<100ug/l se investigó la función tiroidea. De 121 embarazadas la yoduria fue normal en 75, en las que la yoduria matinal no fue diferente a la vespertina (matutina: 305.2 ± 7.0 vs vespertina: 319.2 ± 8.8; NS). No hubo diferencia entre las yodurias de los diversos trimestres. En 46 embarazadas (36.9 por ciento) la yoduria fue baja sin diferencias entre la matutina y vespertina (matutina: 88.12 ± 5.07 µg/l vs 88.7 ± 6.2 µg/l; NS), al igual que entre las yodurias matutinas y vespertinas en los tres trimestres de embarazo. De las embarazadas con yodurias <100ug/l, el 45 por ciento presentó alteraciones de la función tiroidea. No hubo diferencias significativas respecto a la edad entre las embarazadas con baja y normal EUI. La determinación de yoduria matutina y vespertina permitió detectar un mayor número de embarazadas con baja ingesta de yodo y orientó para la búsqueda de disfunción tiroidea que no se hubiera detectado por no contar nuestra Provincia con un Programa de screening para hipotiroidismo gestacional.

The urinary iodine excretion (UIE) assay is an effective method to detect reduced iodine intake. UIE was measured in two different samples (morning and evening) from 121 pregnant women, with a turbid-metric method modified by Pino (normal value =150 ug/l). Furthermore, thyroid function was evaluated in pregnant women with UIE <100 ug/l. From 121 pregnant women, the UIE was normal in 75 with similar morning and evening samples (morning: 305.2 ± 7.0; evening: 319.2 ± 8.8; p: NS). The UIE did no showed differences in different trimesters and in morning and evening samples. The UIE was low in 46 women (36.9 percent), without significant differences between morning and evening (morning: 88.12 ± 5.07 µg/l; evening: 88.7 ± 6.2 µg/l; p: NS). Normal or low UIE were not influenced by the age of pregnant women and 45 percent of pregnant women with UIE <100ug/l showed impaired thyroid function. Morning and evening study of UIE allowed us to detect a higher number of pregnant women with low iodine intake. This study let us to find thyroid function abnormalities likes a screening method, because in our state there is not a public screening program for gestational hypothyroidism.

[An approach to a sanitary and social problem: urinary iodine excretion in pregnant women from a iodine deficient region]. / Un enfoque para un problema sanitario y social: yodurias en embarazadas de una región yododeficiente.

The urinary iodine excretion (UIE) assay is an effective method to detect reduced iodine intake. UIE was measured in two different samples (morning and evening) from 121 pregnant women, with a turbid-metric method modified by Pino (normal value =150 ugl/1). Furthermore, thyroid function was evaluated in pregnant women with UIE <100 ug/l. From 121 pregnant women, the UIE was normal in 75 with similar morning and evening samples (morning: 305.2 +/- 7.0; evening: 319.2 +/- 8.8; p: NS). The UIE did no showed differences in different trimesters and in morning and evening samples. The UIE was low in 46 women (36.9%), without significant differences between morning and evening (morning: 88.12 +/- 5.07 microg/l; evening: 88.7 +/- 6.2 microg/l; p: NS). Normal or low UIE were not influenced by the age of pregnant women and 45% of pregnant women with UIE < 100 ug/l showed impaired thyroid function. Morning and evening study of UIE allowed us to detect a higher number of pregnant women with low iodine intake. This study let us to find thyroid function abnormalities likes a screening method, because in our state there is not a public screening program for gestational hypothyroidism.

Insulin-degrading enzyme hydrolyzes ATP.

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334-341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by (32)P release after hydrolysis of gamma[(32)P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 +/- 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 +/- 3.48 microM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.

The association of acetyl-l-carnitine and nicotinamide remits the experimental diabetes in mice by multiple low-dose streptozotocin.

OBJECTIVES: We studied the effect of acetyl-l-carnitine plus nicotinamide (AC + N) on murine diabetes mellitus induced by multiple low doses of streptozotocin. METHODS: Male C57BL/6J inbred mice were injected intraperitoneally with citrate buffer or streptozotocin (40 mg/kg) for 5 consecutive days, followed by injections of saline solution or AC + N (50 + 25 mg/kg) from days 6 to 110. Four groups were studied: normal control mice (C), treated normal control mice (TC), diabetic mice (D), and treated diabetic mice (TD). TD group was divided into 2 at day 86; treatment was suspended in one group (TDs) and continued in the other until day 110. RESULTS: Weight, plasma glucose, plasma insulin, cellular immune aggression, glucose-stimulated insulin secretion from perifused pancreatic slices, and pancreas histology were studied in each experimental group. Diabetic mice treated with AC + N showed improvements in weight, plasma glucose, and plasma insulin levels without mortality, reaching control values at day 110. Cellular immune aggression and insulin release from pancreatic slices perfusions improved without reaching control values. Histology showed that insulin-immunostained area, the index of insulin immunostained beta cells and beta-cell size, was normalized at the end of the study. CONCLUSIONS: The treatment with AC + N induced remission of autoimmune type 1 diabetes in mice produced by multiple low doses of streptozotocin.

Androgen- and estrogen-dependent regulation of insulin-degrading enzyme in subcellular fractions of rat prostate and uterus.

Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus.

Lipoma in the thyroid?

We report a child with a nodular goiter and a lipoma in the thyroid gland. This association has not been previously described in children. Thyrolipoma and lipomatosis of the thyroid are rare tumors of similar histology and unclear etiology. The lesions and the histology described in this child might represent the beginning of this disease.

Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase.

This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

Sindrome de hipoglucemia autoinmune con anticuerpos especificos anti-insulina humana / Autoimmune hypoglycemia syndrome with specific anti-human insulin antibodies

Se presenta una mujer de 33 años con una historia l1 meses de episodios recurrentes de hipoglucemia severa, asociados a anticuerpos anti-insulina y valores variables de peptido-C. Una extracción ácido-alcohólica de suero mostró un nível basal de insulina de 1.600 uU/ml. La insulina caracterizada por HPLC demostró ser insulina humana. Los anticuerpos fueron específicos para la insulina humana con una subpoblación que reaccionaba con insulina bovina y porcina (IgG, cadena liviana k). Al declinar los síntomas, el tratamiento con plasmaféresis negativizó el título con rapidez. Un seguimiento prolongado demostró la ausencia de recidivas.

Caracterización de sueros antirreceptores de insulina: su aplicabilidad experimental y clínica / Characterization of insulin antireceptors serum: his application experimental and clinic

Se prepararon receptores de hígado de rata por centrifugación diferencial y cromatografía en Sepharosa CL-68 (purificación aproximada 1.000veces) y se inocularon con aquellos 4 conejos. La calidad de los receptores fue controlada por marcación con ATP-Y-32P, electroforesis en poliacrilamida y autorradiografía. Se observó en curvas dosis/respuesta con insulina-125I e IGF-I125I, que el aislamiento usado no excluía el receptor de IGF-I (insulina: 36 fmoles/ml, IGF-I:3,2 fmoles/ml), por lo cual se continuó la purificación en una cromatografía de afinidad de insulina-Sepharosa y posterior marcación con 125I. El antisuero seleccionado C21 demostró una inmunoprecipitación elevada con insulina y baja con IGF-I(insulina: 62,5+4,71%, IGF-I: 24,7+3,16%). En hepatocitos aislados este antisuero no compite con la insulina por su receptor y a una dilución de 1/200 tiene poca capacidad de convertir glucosa-14C a glucógeno-14C, ya que a esa dilución asocia el 50% de los receptores presentes (0,76+0,16 ng/ml, expresado en respuesta biológica equivalente a insulina). para determinar el sitio de unión del antisuero C21 se dirigial receptor de insulina con colagenasa (que digiere específicamente la subunidad B) y se observan estas condiciones la inmunoprecipitibilidad del antisuero C21 cae dramáticamente (receptor de insulina 75,6+2,04%, receptor de insulina digerido: 14,9+1,28%). Se determinó, además, la inmonorreactividad cruzada con receptores humanos para la insulina, observándose un débil cruzamiento a alta concentración (1/75: 1,95+1,32%). El estudio demuestra que el antisueroC21 es predominantemente inmunorreactivo con el receptor de insulina, al que une por su subunidad B, induciendo una escasa respuesta biológica en hepatocitos aislados.

Insulin secretion induced by allogeneic lymphocytis in geneticaly diabetic mice C57BL/KsJ mdb

Se estudió la secreción de insulina y somatostatina (SRIF) inducida por aloantígenos en el ratón genéticamente diabéticos de la cepa C57BL/KsJ mdb-mdb. Ratones diabéticos (db) inyectados con linfocitos alogeneicos (LA) no mostraron ningún incremento en la segunda fase de secreción estimulada por glucosa 27.5 mM y sólo levemente aumentaron su primera fase. Ratones normales inyectados con LA mostraron un incremento significativo en ambas fases de la secreción de insulina estimulada por glucosa 27.5 mM. La secreción de somatostatina de ratones normales o diabéticos inyectados con linfocitos alogeneicos no difirió de aquella obtenida cuando se inyectaron con linfocitos singeneicos. Linfocitos de ratones db inyectados en ratones alogeneicos causaron un incremento en la secreción de insulina que no difirió significativamente de la producida por la inyección de linfocitos alogeneicos de ratones no diabéticos. Linfocitos de ratón diabético inyectados en ratones singeneicos produjeron una secreción de insulina similar a la producida por la inyección de linfocitos de ratones singeneicos no diabéticos, y menor que la producida por la inyección de linfocitos alogeneicos. En resumen, los ratones db mostraron una respuesta hormonal deteriorada al estímulo antigénico, mientras que mantuvieron su acción aloantigénica