Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Growth properties and biochemical characterization of mouse Schwann cells cultured in vitro

Purified secondary cultures of mouse Schwann cells (less than 5% fibroblast contamination) have been obtained by taking advantage of the differential adhesion of Schwann cells and fibroblasts during trypsinization. The growth properties of the purified subcultures changed with time in culture. Cells passaged after 5 days in vitro (DIV) divided rapidly (doubling time 22 h), whereas cells that had been in vitro for longer periods progressively decreased their growth rate, becoming quiescent after 20 or more days. Schwann cells lacked the Thy 1.2 surface antigen, but were positively stained with antigalactocerebroside antibodies after prefixation. Biochemical analyses showed Schwann cells to be enriched in the activities of enzymes characteristic of the myelin-forming cells: 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP), cerebroside sulfotransferase (CST) and UDP-galactose: ceramide galactosyltransferase (CGalT).     

Relation of Cholesterol to Oligodendroglial Differentiation in C-6 Glial Cells

Abstract: The relation of cellular cholesterol content to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial marker enzyme 2′: 3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was determined after alteration of the sterol content of cellular membranes by exposure to compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis. The sterol content and as a consequence, the sterol/phospholipid molar ratio of C-6 glial cells were decreased by treating the cells, in 10% lipoprotein-poor serum, with various concentrations of compactin for 24 h. The degrees of sterol depletion thus produced were maintained for 48 h after removal of the compactin if the cells were maintained in serum-free medium, the culture conditions necessary for induction of CNP in untreated cells. Forty-eight hours after removal of serum, no induction of CNP occurred in cells previously treated with 0.5 μg/ml of compactin, whereas untreated cells exhibited a three- to fourfold increase in CNP activity. Intermediate degrees of sterol depletion resulted in intermediate degrees of inhibition of the CNP induction. Moreover, the morphological expressions of glial differentiation observed in the untreated cells did not occur in the sterol-depleted cells. That the effect of compactin on the induction of CNP relates to depletion of sterol was indicated by the finding that when low-density lipoprotein was added to the compactin-treated cells, the induction of CNP, the morphological expressions of differentiation and the sterol/phospholipid molar ratios were preserved. The degree of sterol depletion that totally prevented the induction of CNP had no effect on (Na⁺+ K⁺)-activated ATPase activity, total protein synthesis and cell viability. The data define a critical role for sterol in oligodendroglial differentiation in this model system.     

Deficiency of immunoreactive somatostatin in the median eminence of Snell dwarf mice

Snell dwarf mice (dw/dw) are characterized by a genetically determined, congenital lack of pituitary GH, TSH and prolactin. Given that hypothalamic somatostatin is involved in the regulation of pituitary GH and TSH release, it was decided to investigate the content of immunoreactive somatostatin (IRS) in the median eminence of dw/dw and phenotypically normal mice of the same strain. The content of IRS in the pyloric antrum and pineal gland of these animals was also examined. The effects of ovariectomy and of hyperprolactinemia (induced by a pituitary graft under the kidney capsule) on the median eminence content of IRS were also studied in both normal and dwarf mice. Median eminence IRS content was significantly lower in the dw/dw (23.6 +/- 1.8 ng) than in normal mice (57.4 +/- 7.1 ng); no difference was found in the pyloric IRS content of dw/dw (16.9 +/- 1.6 ng/mg of protein) and normal animals (13.8 +/- 1.9 ng/mg of protein), nor in the pineal content of IRS (639.4 +/- 64.4 pg/gland in the dw/dw; 732 +/- 265 pg/gland in normals). Neither ovariectomy nor hyperprolactinemia were found to affect the IRS content in the tissues studied in normal or dwarf mice. Treatment of an additional group of 9 dwarf mice with L-thyroxine (L-T4 2 micrograms/48 h. s.c. for 2 weeks) significantly increased the animals weight (10.2 +/- 0.4 g versus 7.4 +/- 0.3 g) and produced maturation of facial features; however, it did not change the IRS content in any of the tissues studied. It is concluded that the content of IRS in the median eminence of mice with a congenital lack of GH, TSH and prolactin is significantly reduced and that this is unlikely to be related to the deficiency of thyroid hormones in these animals.     

Effect of prolactin and growth hormone on prolactin and LH receptors in the dwarf mouse.

Dwarf mice (DW/J;dw/dw) which exhibit a deficiency of prolactin and GH secretion were treated for 8 days with ovine prolactin and/or human GH (10 or 20 mug/day) and the effect on hepatic and testicular prolactin receptors was investigated. In both sexes there was a significant increase in body weight after all hormone treatments, but an increment in testicular weight was observed only after prolactin administration. Prolactin treatment increased the specific binding % of prolactin in liver membranes in females but not males, and in testicular homogenates (together with an increase in LH receptors). The results suggest that lack of prolactin but not of GH retards sexual development in these mice. Treatment with prolactin partly counteracts this deficiency, and the effect may be mediated by the induction of hepatic and testicular prolactin and LH receptors.     

Pituitary and serum concentrations of prolactin and GH in Snell dwarf mice.

Secretions of prolactin and GH in dwarf mice were studied using homologous radioimmunoassays. Blood samples from adult male and femal dw/dw mice were collected by orbital puncture and by decapitation. Compared to related normals (+/?), pituitary concentrations of GH and prolactin were very low in dw/dw mice, the concentrations of prolactin being more scarce than those of GH. Prolactin and GH concentrations were also lower in sera of dw/dw mice, but the relative differences appeared sex-dependent: serum GH was more reduced in males than in females while serum prolactin was more depressed in females. The data confirm earlier indications of deficiencies in the circulating levels of prolactin and GH in dwarf mice and suggest that the hypoactivity of these hormones may be crucial to some of the anomalies found in this mutant.     

Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Interaction of Porcine Growth Hormone and Thyroxine

HAUSMAN, D.B., G.J. HAUSMAN, AND R.J. MARTIN. Endocrine regulation of fetal adipose tissue metabolism in the pig: interaction of porcine growth hormone and thyroxine. Obes Res. 1999;7:76–82. Objective : This study tested the hypothesis that combined treatment of thyroxine (T₄) and growth hormone (GH) could normalize cellular and metabolic aspects of adipose tissue development of hypophysectomized fetal pigs. Research Methods and Procedures : On day 70 of gestation, pig fetuses were hypophysectomized by microcauterization or remained intact. Hypophysectomized fetuses remained untreated or were treated from day 90 to day 105 of gestation with T₄, GH, or a combination of both hormones. Results : Body weights were unaffected by hypophysectomy or hormone treatment. De novo lipogenesis in subcutaneous adipose tissue was increased 10-fold by hypophysectomy, consistent with our previous results. This increase was abolished by GH treatment in the hypophysectomized fetuses. In contrast, T₄ treatment of the hypophysectomized fetuses resulted in a 12-fold further increase in adipose tissue lipogenesis, an effect that was negated by concomitant administration of GH. Lipolytic response to isoproterenol was decreased by hypophysectomy, unaffected by GH treatment, and restored to intact values by T₄ or by T₄+GH treatment in the hypophysectomized fetuses. Discussion : In contrast to T₄, GH does not influence serum insulin-like growth factor-I or adipose tissue lipolysis, but decreases lipogenesis in the fetal pig. However, replacing both T₄ and GH normalized hypophysectomized fetuses to a greater extent than either GH or T₄ alone. Thus, any influence of thyroid hormones on stimulating adipose tissue lipogenesis in the developing fetal pig may be normally counterregulated by pituitary-derived growth hormone.     

Adiposity profile in the dwarf rat: an unusually lean model of profound growth hormone deficiency

This study describes the previously uncharacterized ontogeny and regulation of truncal adipose reserves in the profoundly GH-deficient dwarf (dw/dw) rat. We show that, despite normal proportionate food intake, dw/dw rats develop abdominal leanness and hypoleptinemia (circulating leptin halved in dw/dw males, P < 0.05) during puberty. This contrasts with the hyperleptinemia seen in moderately GH-deficient Tgr rats (circulating leptin doubled at 6 wk of age, P < 0.05) and in GH receptor-binding protein (GHR/BP)-null mice (circulating leptin doubled; P < 0.05). This lean/hypoleptinemic phenotype was not completely normalized by GH treatment, but dw/dw rats developed abdominal obesity in response to neonatal MSG treatment or maintenance on a high-fat diet. Unlike Tgr rats, dw/dw rats did not become obese with age; plasma leptin levels and fat pad weights became similar to those in wild-type rats. In contrast with truncal leanness, tibial marrow adiposity was normal in male and doubled in female dwarves (P < 0.01), this increase being attributable to increased adipocyte number (P < 0.01). Neonatal MSG treatment and high-fat feeding elevated marrow adiposity in dw/dw rats by inducing adipocyte enlargement (P < 0.05). These results demonstrate that, despite lipolytic influence of GH, severe GH deficiency in dw/dw rats is accompanied by a paradoxical leanness. This lean/hypoleptinemic phenotype is not solely attributable to reduced GH signaling and does not appear to result from a reduction in nutrient intake or the ability of dw/dw adipocytes to accumulate lipid. Disruption of preadipocyte differentiation or adipocyte proliferation in the dw/dw rat may lead to the development of this unusually lean/hypoleptinemic phenotype.     

Kallikrein-binding protein is induced by growth hormone in the dwarf rat.

Rat kallikrein-binding protein (KBP), a member of the serpin family, is a tissue kallikrein inhibitor. It has been shown to be a potential pathogenic factor of diabetic retinopathy and may play a role in animal development and growth. To determine whether reduced KBP expression is involved in retarded animal growth, we examined the in vivo effect of growth hormone (GH) deficiency on the expression of KBP in the Lewis dwarf (dw/dw). We found that serum levels of functionally active KBP were reduced in the dwarf rat (P < 0.05) as determined by complex formation assay between serum KBP and (125)I-labeled rat tissue kallikrein. Enzyme-linked immunosorbent assay showed that KBP levels were significantly reduced in the serum of the dwarf rat compared to the Lewis rat (213.8 ng/ml vs. 413.8 ng/ml, n = 4, P < 0.01). The decreased KBP levels were confirmed by Western blot analysis. Moreover, treatment of the dwarf rat with recombinant human GH for 4 wk resulted in a significant increase in KBP activity (P < 0.01) and serum KBP levels compared with the untreated dwarf rat (549.8 ng/ml, n = 5, vs. 213.8 ng/ml, n = 4, P < 0.02). Northern blot analysis and densitometry showed that liver KBP mRNA levels were reduced by fivefold in the dwarf rat compared to the Lewis rat and the decrease was reversed by the GH treatment. These results indicate that the KBP levels are regulated at the RNA level. Furthermore, in vitro studies using cultured rat hepatocytes showed that GH may have a direct regulatory effect on KBP expression since KBP levels increased in the conditioned media of cells treated with GH. These results demonstrated that KBP is reduced in the genetic dwarf rat and is restored to normal by GH; therefore, KBP is a GH-dependent protein and may be a new target for studying the mechanism of pathological animal growth.     

CERAMIDE-GALACTOSYLTRANSFERASE AND CEREBROSIDE-SULPHOTRANSFERASE LOCALISATION IN GOLGI MEMBRANES ISOLATED BY A CONTINUOUS SUCROSE GRADIENT OF MOUSE BRAIN MICROSOMES

Abstract— A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.11), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.     

Impaired peripheral T3 production but normal induced thyroid hormone secretion in the sex-linked dwarf chick embryo

Plasma concentrations of thyroxine (T4), triiodothyronine (T3) and chicken GH (cGH), together with hepatic 5'-monodeiodination (5'-D) activity, were measured in normal (Dw) and dwarf chick (dw) embryos at incubation d 18. An injection of 10 micrograms of ovine GH (oGH) raised plasma concentrations of T3 in Dw embryos after 1 and 2 h and stimulated hepatic 5'-D activity after 2 h. A non-specific increase in T4 was also observed after 1 h in Dw animals probably due to the heterologous nature of the injection. These effects were not observed in dw embryos. An injection of 1 microgram of TRH was able to increase cGH levels after 15 min in Dw embryos, whereas the the observed increase in the dw group was not significant. In Dw embryos, 0.01, 0.1 and 1 microgram of TRH increased plasma concentrations of T3 in a dose-dependent way, whereas in dw embryos, no reaction to the TRH injections was seen, except for the highest dose used. Contrary to this observation, T4 was increased to the same level in both Dw and dw embryos following TRH injections. An injection of 1 microgram of ovine CRH increased corticosterone after 0.5 h and elevated T3 and T4 after 2 h to the same extent in Dw and dw embryos. It is concluded that the thyrotrophic activities of TRH and oCRH and the corticotropic activity of oCRH do not differ between normal and sex-linked dwarf embryos. However TRH and GH were unable to stimulate the T4-T3 conversion in the liver of dw embryos, presumably due to the lack of hepatic GH receptors in these animals.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland

Summary The effects of 5-dihydrotestosterone (DHT) and thyroxine (T₄) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administraition of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T₄ increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T₄ in adrenalectomized mice suggests that T₄ has a direct effect on the submandibular gland.     

Selenium content of livers from sex-linked dwarf and normal broiler breeders

Plasma concentrations of cGH, T₃, and T₄ were not different between dwarf and normal broiler breeders. Normal hens had a liver selenium content of 710±35 ng/g, and dwarf hens 656 ±nine ng/g (n=8). Following injections into a wing vein of different doses (1.5, 3, 6, 12, and 24 μg/kg) of the hypothalamic hormone TRH, GH was increased after 15 min. This effect seemed to last longer in dwarf chickens. Plasma concentrations of T₃ increased significantly 1 h after TRH in normal hens, but TRH was ineffective in raising T₃ levels in dwarf animals. The selenium content of livers obtained following decapitation after 2 h was also increased in normal hens up to 902±42 ng/g using the highest dose of TRH (24 μg/kg). This seemed not to be the case for dwarf animals. A much smaller. number of hepatic cGH receptors was also found in dwarf hens, whereas the affinity of the hepatic GH receptor was not influenced by the genotype. It is concluded that the sex-linked dwarf hens are unable to increase their hepatic T₄ into T₃ conversion following a TRH challenge probably because of a deficiency in hepatic GH receptors. The lower content of selenium in dwarfs and their inability to increase its uptake after TRH seem therefore to support the hypothesis that selenium has a direct role in the activity of the 5′-deiodinase complex.     

The effect of high temperatures on tissue lactate,pyruvate and venous blood properties in the rat

Brain, muscle and liver pyruvate and L(+) lactate were determined in rats (1) control, T_re=37.2°C; (2) thermal stress T_re=40.8°C; (3) terminal thermal stress T_re=43.8°C, and (4) hypoxic death at reduced oxygen tension. A second experiment was conducted to examine venous blood acid-base parameters in heated rats at imminent death. Group 2 failed to show a significant change in brain lactate concentration, but muscle lactate decreased and liver lactate increased significantly (27 and 48%, respectively). Group 3 showed significant increases of 65 and 125% in the lactate content of brain and liver, respectively, but in this instance no significance was attributed to the 12% decrease in muscle lactate. Hypoxia in Group 4 resulted in the greatest increases in tissue lactate in brain, muscle and liver (130, 50 and 171%, respectively). The pyruvate concentrations of brain and liver in Group 2 exhibited no change, but muscle pyruvate decreased significantly (59%). Group 3 brain and muscle pyruvate decreased significantly by 57 and 74%, while liver pyruvate remained unchanged. Hypoxia (4) produced no significant differences in pyruvate levels from those observed in Group 3. The changes in venous blood properties of rats heated until respiratory movement ceased suggested acute and severe metabolic acidosis while animals exhibiting heat induced spasms and loss of coordination showed only slight decreases in blood pH and bicarbonate levels. Brain water content did not change, but muscle was dehydrated and liver tissue water content increased in rats exposed to lethal temperatures. The results indicate that hypoxia probably occurs in rat tissues at high temperatures, but not to a degree that would result in death.

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Zusammenfassung Gehirn, Muskel und Leberpyruvat and 1-Laktat wurden bestimmt in Ratten (1) Kontrollen, T_re=37,2°C; (2) Hitze T_re=40,8°C; (3) tödliche Belastung T_re=43,8°C und (4) Tod durch Hypoxie bei reduziertem PO₂. In einem zweiten Experiment wurde der Säure Basengehalt des venösen Blutes in erhitzten Tieren vor Eintritt des Todes bestimmt. Gruppe 2 zeigte keine signifikante Änderung in der Gehirnlaktatkonzentration, dagegen fiel das Muskellaktat (–27%) und das Leberlaktat stieg significant(+48%). Gruppe 3 zeigte einen signifikanten Anstieg des Gehirn- (+65%) und Leberlaktats (+125%), während das Muskellaktat fiel (–11%). Hypoxie in Gruppe 4 bewirkte den grössten Anstieg des Laktats im Gehirn (+130%), Muskel (+50%) und Leber (+171%). Die Pyruvatkonzentration in Gruppe 2 zeigte nur im Muskel einen Anstieg auf +59%. In Gruppe 3 fielen das Gehirn- (–57%) und Muskelpyruvat (–74%) signifikant, ohne Änderung in der Leber. Ähnliche Resultate ergaben sich bei Hypoxie in Gruppe 4. Vor dem Eintritt des Hitzetodes war im Blut hochgradige Acidose nachweisbar, während Tiere mit Hitzespasmus und Koordinationsstörungen nur gering niedrigere pH- und Bikarbonatwerte aufwiesen. Der Gehirnwassergehalt blieb unverändert, der Muskel war dehydriert und die Leber zeigte einen höheren Wassergehalt bei letal hohen Temperaturen. Die Ergebnisse zeigen, dass Hypoxia bei Hyperthermie auftritt, doch nicht in dem Ausmasse, um zum Tod zu führen.

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Resume On a déterminé les pyruvates et lactates du cerveau, des muscles et du foie de rats présentant 4 particularités différentes: (1) T_re (température rectale) = 37,2°C (contrôle); (2) T_re = 40,8°C (contrainte de chaleur); (3) T_re = 43,8°C (contraite de chaleur limite) et (4) morts par hypoxie par suite de la réduction de la pression partielle de l'oxygène. Dans un second essai, on a déterminé le rapport acides/bases dans le sang veineux de rats sous contrainte de chaleur, juste avant leur trépas. Dans le groupe 2, on n'a pas trouvé de modification significative de la concentration des lactates du cerveau. Par contre, celle des muscles a diminué (–27%) et celle du foie a augmenté (+48%) et cela de façon significative. Dans le groupe 3, on constate une hausse du taux de lactates dans le cerveau (+65%) et dans le foie (+125%), alors que celui des muscles diminue (–11%). L'hypoxie du groupe 4 a provoqué une forte augmentation des lactates aussi bien dans le cerveau (+130%), les muscles (+50%) que le foie (+171%). La concentration des pyruvates ne fut sensiblement modifiée dans le groupe 2 que pour les muscles (+59%). Dans le groupe 3, on en note une diminution significative dans le cerveau (–57%) et dans les muscles (–74%) alors que le taux du foie reste inchangé. On a obtenu des résultats analogues par l'hypoxie dans le groupe 4. On a pu constater une acidose aigue du sang juste avant la mort de chaleur, alors que les animaux atteints de spasmes et de perturbations dans la coordination ne présentaient que peu de variations du pH et du taux de bicarbonate. Dans un état létal par hautes températures, la teneur en eau du cerveau est restée inchangée. Les muscles étaient par contre déshydratés et le foie contenait davantage d'eau. Ces résultats ont montré que, lorsque la température augmente, les animaux souffrent d'hypoxie, mais pas au point d'en mourir.

     

Cerebral myelinogenesis in theSnell dwarf mouse: Stimulatory effects of GH and T4 restricted to the first 20 days of postnatal life

We attempted to define the critical time period during early postnatal life when GH and T₄ are essential for myelination. We administered bGH and T₄ toSnell dwarf mice during the first and second 20 days after birth. Positive results were obtained only when hormones were given during the first 20 days of postnatal life. We observed a distinct increase in brain weight, DNA content, CNPase activity and a remarkably increased level of spontaneous locomotion activity with a diurnal periodicity. Morphological observations of brain sections stained for myelin basic protein (MBP) correlated the biochemical findings. The later administration of hormones was ineffective. Our interpretation is that the administration of exogenous hormones led to increased myelinogenesis through their stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.     

Isolated deficiency of immunocytochemically detected somatostatin in Snell dwarf, but not in “little,” mice

Immunocytochemical staining of the neuropeptide somatostatin was evaluated in the brains of two growth hormone-deficient mouse mutants, Snell dwarf (dw/dw), and “little” (lit/lit). In Snell dwarf mice, in which GH is undetectable, an isolated somatostatin deficiency was observed in hypothalamic median eminence and in anterior periventricular hypothalamic neurons which are afferent to median eminence, compared to somatostatin staining in normal mice of the same strain (DW/?). Somatostatin staining in all other CNS areas in dwarfs was identical to that in normal mice. In contrast, in little mice, which exhibit 5–10% of normal GH levels, somatostatin expression was comparable between mutants and normal mice (LIT/?) in all CNS areas, including median eminence-afferent neurons in anterior periventricular hypothalamus. The results suggest that expression of somatostatin in hypophysiotropic CNS is dependent upon minimal pituitary GH secretion.     

Effect of thyroid hormone administration on the depletion of circulating glutathione in the isolated perfused rat liver and its relationship to basolateral γ-glutamyltransferase activity

The influence of thyroid hormone administration on liver glutathione (GSH) extraction in the isolated perfused liver was studied in fed rats for a period of 1–7 days following a single dose of 0.1 mg 3,5,3′-triiodothyronine (T₃)/kg. T₃ treatment led to an early and transient calorigenic response, as well as an enhancement in liver GSH removal, reaching a maximal effect at 2 days after hormone administration, which was normalized in the 3- to 7-day period studied. Addition of the γ-glutamyltransferase (γ-GT) inhibitor DL-serineborate (4 mM) to the perfusate abolished the increase in the hepatic removal of GSH elicited by T₃, and enhanced the sinusoidal concentration of GSH, studied at 2 days after hormone administration. These data support the role of hepatic basolateral γ-GT ectoactivity in the depletion of portally added and liver-derived GSH as an adaptive response to recover GSH levels after reduction by T₃-induced oxidative stress.     

Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats

The purpose of this study was to investigate the possible antioxidant effect of an aqueous extract of Ajuga iva (Ai) in streptozotocin (STZ)-induced diabetic rats. Twelve diabetic rats were divided into two groups fed a casein diet supplemented or not with Ai (0.5%), for 4 weeks. In vitro, the Ai extract possessed a very high antioxidant effect (1 mg/ml was similar to those of trolox 300 mmol/l). The results indicated that plasma thiobarbituric acid reactive substances (TBARS) values were reduced by 41% in Ai-treated compared with untreated diabetic rats. TBARS concentrations were lower 1.5-fold in liver, 1.8-fold in heart, 1.9-fold in muscle and 2.1-fold in brain in Ai-treated than untreated group. In erythrocytes, Ai treatment increased significantly the activities of glutathione peroxidase (GSH-Px) (+25%) and glutathione reductase (GSSH-Red) (+22%). Superoxide dismutase activity was increased in muscle (+22%), while GSH-Px activity was significantly higher in liver (+28%), heart (+40%) and kidney (+45%) in Ai-treated compared with untreated group. Liver and muscle GSSH-Red activity was, respectively, 1.6- and 1.5-fold higher in Ai-treated than untreated diabetic group. Catalase activity was significantly increased in heart (+36%) and brain (+32%) in Ai-treated than untreated group. Ai treatment decreased plasma nitric oxide (?33%), carbonyls (?44%) and carotenoids (?68%) concentrations. In conclusion, this study indicates that Ajuga iva aqueous extract improves the antioxidant status by reducing lipid peroxidation and enhancing the antioxidant enzymes activities in plasma, erythrocytes and tissues of diabetic rats.     

Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers

Three triazole-linked nonionic xylo-nucleoside dimers T^L-t-T^xL, T^L-t-A^BzxL and T^L-t-C^BzxL have been synthesized for the first time by Cu(I) catalyzed azide-alkyne [3 + 2] cycloaddition reaction (CuAAC) of 1-(3′-azido-3′-deoxy-2′-O,4′-C-methylene-β-D-ribo-furanosyl)thymine with different alkynes, i.e., 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)thymine, 9-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylo-furanosyl)-N6-benzoyladenine and 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)-N4-benzoylcytosine in 90%–92% yields. Among the two Cu(I) reagents, CuSO₄.5H₂O-sodium ascorbate in THF:^tBuOH:H₂O (1:1:1) and CuBr.SMe₂ in THF used for cycloaddition (click) reaction, the former one was found to be better yielding than the latter one.     

Increased Oxidative Metabolism and Neurotransmitter Cycling in the Brain of Mice Lacking the Thyroid Hormone Transporter Slc16a2 (Mct8)

Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T₄ and T₃) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T₄ and T₃. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-¹³C) glucose and brain extracts prepared and analyzed by ¹³C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T₃ by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.