Grape seed procyanidin extract inhibits adipogenesis and stimulates lipolysis of porcine adipocytes in vitro.

The objective of this article was to evaluate in vitro effect of grape seed procyanidin extract (GSPE) on differentiation, proliferation, and lipolysis of porcine adipocytes, providing a molecular basis for the use of GSPE in pig fat regulation. Primary preadipocytes isolated from subcutaneous adipose tissue of pigs were used as the in vitro cell model. Treatment of GSPE repressed preadipocyte differentiation, as evidenced by reduced lipid accumulation, decreased mRNA expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4), as well as enhanced expressions of preadipocyte factor-1. Activity of glycerol-3-phosphate dehydrogenase (GPDH), one of the most important enzymes in the pathway for triacylglycerol biosynthesis, was also decreased. Furthermore, GSPE could suppress preadipocyte proliferation by inducing G0/G1 cell cycle arrest and cell apoptosis. In porcine mature adipocytes, treatment with GSPE attenuated lipid content and GPDH activity, and the release of both free fatty acid and glycerol were enhanced; mRNA expressions of key lipolytic transcription factors, including hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), were elevated in GSPE-treated adipocytes. In summary, our results suggest GSPE inhibits porcine preadipocyte differentiation and proliferation and stimulates lipolysis of mature adipocytes, thus providing novel insights for further exploring the use of GSPE as a fat accumulation inhibitor.

Molecular action of metformin in hepatocytes: an updated insight.

Although, metformin is a drug of the first choice in the treatment of type 2 diabetes mellitus, its molecular action is not fully determined. It is widely accepted that the antihyperglycemic effect of metformin is a result of a decrease in hepatic glucose production, and several cellular targets of the drug have been proposed. The reduction of gluconeogenesis evoked by metformin may be a result of an energy deficit evoked through the inhibition of mitochondrial respiratory chain complex I and/or increased cytosolic redox state and decreased mitochondrial redox state elicited by the inhibition of mitochondrial glycerophosphate dehydrogenase (mGPD). Metformin mediated reduction of hepatic gluconeogenesis was found to be AMP-activated protein kinase (AMPK) dependent and independent, including the inhibition of gluconeogenesis gene expression and allosteric regulation of key gluconeogenesis enzymes. Recently, it was reported that inhibition of mGPD by metformin decreases the level of dihydroxyacetone phosphate and reduces the conversion of lactate to pyruvate, that in consequence diminishes the utilization of glycerol and lactate for gluconeogenesis. The purpose of this paper is to discus molecular mechanisms responsible for the metabolic action of metformin.

In-silico Leishmania target selectivity of antiparasitic terpenoids.

Neglected Tropical Diseases (NTDs), like leishmaniasis, are major causes of mortality in resource-limited countries. The mortality associated with these diseases is largely due to fragile healthcare systems, lack of access to medicines, and resistance by the parasites to the few available drugs. Many antiparasitic plant-derived isoprenoids have been reported, and many of them have good in vitro activity against various forms of Leishmania spp. In this work, potential Leishmania biochemical targets of antiparasitic isoprenoids were studied in silico. Antiparasitic monoterpenoids selectively docked to L. infantum nicotinamidase, L. major uridine diphosphate-glucose pyrophosphorylase and methionyl t-RNA synthetase. The two protein targets selectively targeted by germacranolide sesquiterpenoids were L. major methionyl t-RNA synthetase and dihydroorotate dehydrogenase. Diterpenoids generally favored docking to L. mexicana glycerol-3-phosphate dehydrogenase. Limonoids also showed some selectivity for L. mexicana glycerol-3-phosphate dehydrogenase and L. major dihydroorotate dehydrogenase while withanolides docked more selectively with L. major uridine diphosphate-glucose pyrophosphorylase. The selectivity of the different classes of antiparasitic compounds for the protein targets considered in this work can be explored in fragment- and/or structure-based drug design towards the development of leads for new antileishmanial drugs.

Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria.

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.

Regulation of differentiating pig preadipocytes by retinoic acid.

All-trans retinoic acid (ATRA) potently inhibits the differentiation of porcine preadipocytes in primary culture; however, the mechanism by which ATRA exerts this effect in pigs is poorly understood. The objective of this study was to use retinoid receptor-specific ligands to investigate the mechanism underlying the antiadipogenic action of retinoids in cultured pig preadipocytes by identifying the retinoid receptor mediating this action and examining the effect of retinoids on the expression of key adipogenic transcription factors. Stromal-vascular cells were harvested from porcine adipose tissue and cultured in serum-free medium. Glycerol-3-phoshphate dehydrogenase (GPDH) activity, a late marker of preadipocyte differentiation, was decreased (P < 0.01) by the addition of 0 to 10 microM of either ATRA, a nonspecific agonist for both the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) or the selective RAR agonist, 4-(E-2-[5,6,7,8-tet-rahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB). Addition of increasing amounts of Ro-61, a RAR-specific antagonist (0 to 10 microM) prevented ATRA and TTNBP from decreasing GPDH activity. Addition of methoprene acid, an RXR-specific agonist, increased (P < 0.01) GPDH activity. Preadipocytes were then continuously treated with 10 nM of TTNPB in the presence or absence of 1 microM Ro-61, and mRNA was isolated on d 2 and 8. Addition of TTNPB decreased (P < 0.001) the expression of peroxisome proliferator-activated receptor gamma (PPARgamma), sterol regulatory element-binding protein-1c (SREBP-1c), retinoid X receptor alpha (RXRalpha), and adipocyte fatty acid binding protein (aP2) mRNA transcripts, whereas these effects were prevented by the presence of Ro-61. Interestingly, TTNBP increased (P < 0.001) the mRNA abundance of the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1), whereas Ro-61 prevented this increase. These changes were independent of alterations in the mRNA abundances of the retinoic acid receptor alpha, and CCAAT/enhancer binding protein alpha and beta (C/EBPbeta; C/EBPalpha) genes. These results indicate that retinoic acid inhibits porcine preadipocyte differentiation by a mechanism that involves activation of the RAR and downregulation of PPARgamma, RXRalpha, and SREBP-1C mRNA. This mechanism is independent of changes in C/EBPbeta and C/EBPalpha mRNA abundance and may involve COUP-TF.

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria.

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.

Vitamin C is one of the lipolytic substances in green tea.

We have studied the influence of vitamin C contained in green tea on the lipolysis of well-differentiated 3T3-L1 cells. When mature adipocytes were exposed to vitamin C the triglyceride concentration was decreased (p < 0.05) and the activity of glycerophosphate dehydrogenase, a marker of adipose conversion, was significantly inhibited (p < 0.01). These data suggest that green tea may have a lipolytic activity due to the mechanism by which the vitamin C contained in it inhibits triglyceride accumulation.

1alpha,25-Dihydroxyvitamin D3 modulates human adipocyte metabolism via nongenomic action.

We reported recently that suppression of the renal 1alpha,25-dihyroxyvitamin D3 (1lpha,25-(OH)2-D3) production in aP2-agouti transgenic mice by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, inhibits lipogenesis, and reduces adiposity. However, it was not clear whether this modulation of adipocyte metabolism by dietary calcium is a direct effect of inhibition of 1alpha,25-(OH)2-D3-induced [Ca2+]i. Accordingly, we have now evaluated the direct role of 1alpha,25-(OH)2-D3. Human adipocytes exhibited a 1alpha,25-(OH)2-D3 dose-responsive (1-50 nM) increase in [Ca2+]i (P<0.01). This action was mimicked by 1alpha,25-dihyroxylumisterol3 (1alpha,25-(OH)2-lumisterol3) (P<0.001), a specific agonist for a putative membrane vitamin D receptor (mVDR), and completely prevented by 1b,25-dihydroxyvitamin D3 (1beta,25-(OH)2-D3), a specific antagonist for the mVDR. Similarly, 1alpha,25-(OH)2-D3 (5 nM) caused 50%-100% increases in adipocyte fatty acid synthase (FAS) expression and activity (P<0.02), a 61% increase in glycerol-3-phosphate dehydrogenase (GPDH) activity (P<0.01), and an 80% inhibition of isoproterenol-stimulated lipolysis (P<0.001), whereas 1beta,25-(OH)2-D3 completely blocked all these effects. Notably, 1alpha,25-(OH)2-lumisterol3 exerted more potent effects in modulating adipocyte lipid metabolism, with 2.5- to 3.0-fold increases in FAS expression and activity (P<0.001) and a threefold increase in GPDH activity (P<0.001). Also 1alpha,25-(OH)2-lumisterol3 was approximately twice as potent in inhibiting basal lipolysis (P<0.025), whereas 1beta,25-(OH)2-D3 completely blocked all these effects. These data suggest that 1alpha,25-(OH)2-D3 modulates adipocyte Ca2+ signaling and, consequently, exerts a coordinated control over lipogenesis and lipolysis. Thus, a direct inhibition of 1alpha,25-(OH)2-D3-induced [Ca2+]i may contribute to an anti-obesity effect of dietary calcium, and the mVDR may represent an important target for obesity.

Histamine N-methyltransferase regulates histamine-induced phosphoinositide hydrolysis in guinea pig cerebellum.

We report here that the dose-response curve of the histamine-stimulated phosphoinositide hydrolysis in the guinea pig cerebellar slices was shifted to the left when the slices were pretreated with SKF 91488 (100 microM), a specific inhibitor of histamine N-methyltransferase (HMT). In contrast, the pretreatment of the cerebellar slices with aminoguanidine (100 microM - 1 mM), an inhibitor of diamine oxidase, had no effect on histamine-induced phosphoinositide hydrolysis. HMT mRNA was expressed abundantly in cerebellum, especially in Purkinje cells. These observations suggest that HMT regulates histaminergic neurotransmission in guinea pig cerebellum more predominantly than diamine oxidase in histamine degradation.

Ergosteroids IV: synthesis and biological activity of steroid glucuronosides, ethers, and alkylcarbonates.

The 7-oxo derivative of dehydroepiandrosterone is more active than the parent steroid and is devoid of adverse side effects in rats, monkeys and humans. In anticipation of possible therapeutic use we have sought more active, longer lasting forms of 7-oxo- and 7beta-hydroxydehydroepiandrosterones. The 7-oxo- and 7-hydroxy steroids have been converted to glucuronides, ethers and carbonate esters. The syntheses of these compounds are described and their ability to induce the formation of liver thermogenic enzymes when fed to rats is reported. Some of the new derivatives were found to be somewhat more effective than the equimolar amounts of 7-oxo-DHEA with which they were compared in each experiment.

Inhibition of preadipocyte differentiation by myostatin treatment in 3T3-L1 cultures.

Myostatin, a new TGF-beta family member, is known as a muscle growth inhibitor, but its role in adipocyte development has not been studied. To test the role of Myostatin in 3T3-L1 preadipocyte differentiation, we treated cultured 3T3-L1 preadipocytes with Myostatin dissolved in 0.1% trifluoroacetic acid (TFA) during differentiation after they had become confluent. Myostatin treatment significantly decreased glycerol-3-phosphate dehydrogenase (GPDH) activity and oil Red-O staining compared to controls that did not receive Myostatin. Western blot analysis showed that the expression levels of CCAAT/enhancer binding protein alpha (C/EBP alpha) and peroxisome proliferator-activated receptor gamma (PPAR gamma) were significantly decreased by Myostatin treatment (P < 0.05). However, the expression of C/EBP beta was not significantly changed by the treatment (P > 0.05). From RT-PCR result, the relative level of leptin mRNA in Myostatin-treated cells was not significantly different (P > 0.1) from the level in cells without Myostatin treatment. Our data show that Myostatin, a secreted protein from muscle, inhibits preadipocyte differentiation in 3T3-L1 cells, which is mediated, in part, by altered regulation of C/EBP alpha and PPAR gamma.

Inhibition of lipogenesis by pycnogenol.

The influence of pycnogenol on the adipose conversion of 3T3-L1 cells by insulin was studied. In week 3 of culture with insulin, pycnogenol was found to inhibit significantly the expression of glycerophosphate dehydrogenase (p < 0.01). This finding suggests that pycnogenol inhibits the accumulation of lipid droplets in adipose tissue.

Effect of hibernation, thyroid hormones and dexamethasone on cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase from jerboa (Jaculus orientalis).

Tissue distribution of the cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (cGPDH and mGPDH) activities in jerboa (Jaculus orientalis), a hibernator, shows the highest level of enzyme activity in skeletal muscle and brown adipose tissue, respectively. The effect of hibernation on cGPDH indicates an increase of activity in all tissues examined. In contrast, hibernation decreases mGPDH activity in all tissues, except skeletal muscle. The effect of thyroid hormones on GPDH activity was tissue specific: in kidneys, cGPDH activity doubled in euthermic jerboas treated with T4. In contrast, 6-n-propyl-2-thiouracil treatment provokes an increase of enzyme activity in brown adipose tissue, liver and brain. T4 treatment leads to a 2.7-fold increase in liver mGPDH activity. 6-n-propyl-2-thiouracil treatment decreases mGPDH activity in the skeletal muscle whereas the opposite effect was observed in brain. Dexamethasone stimulates cGPDH in all tissues examined, except skeletal muscle and kidneys. In the case of mGPDH activity, this increase was observed only for brown adipose tissue and brain. Our results suggest that hibernation, thyroid hormones and dexamethasone probably play a role in the regulation of cGPDH and mGPDH activities in jerboa. Our findings confirm that these enzymes are involved in metabolic adaptation to thermal stress in Jaculus orientalis.

Vitamin B12-induced alterations in activities of alpha-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch).

Different doses of vitamin B12 (0.25, 0.5, 1, 2 and 4 micrograms/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-alpha-glycerophosphate dehydrogenase (alpha-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The alpha-GPD activity increased at doses of 0.5, 1, 2 and 4 micrograms/g vitamin B12. A dose of 0.5 microgram/g vitamin B12 induced less activity than higher doses. ME activity increased with 1, 2 and 4 micrograms/g of vitamin B12/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B12 administration. Cycloheximide treatment inhibited the vitamin B12-induced increase in alpha-GPD and ME activity. Thus, vitamin B12 is involved in the induction of some enzymes in fish brain.

Role of the sulfonylurea receptor in regulating human adipocyte metabolism.

A regulatory role for intracellular Ca2+ ([Ca2+]i) in adipocyte lipogenesis, lipolysis and triglyceride accumulation has been demonstrated. Compounds acting on the pancreatic sulfonylurea receptor (SUR) to increase (e.g., glibenclamide) or decrease (e.g., diazoxide) [Ca2+]i cause corresponding increases and decreases in weight gain. However, these weight gain and loss effects have been attributed to insulin release rather than to the primary effects of these compounds on the adipocyte SUR and its associated K(ATP) channel. Accordingly, we have evaluated the direct role of the human adipocyte SUR in regulating adipocyte metabolism. We used RT-PCR with primers for a highly conserved region of SUR1 to demonstrate that human adipocytes express SUR1. The PCR product was confirmed by sequence analysis and used as a probe to demonstrate adipocyte SUR1 expression by Northern blot analysis. Adipocytes exhibited glibenclamide dose-responsive (0-20 microM) increases in [Ca2+]i (P<0.05). Similarly, glibenclamide (10 microM) caused a 67% increase in adipocyte fatty acid synthase activity (P<0.001), a 48% increase in glycerol-3-phosphate dehydrogenase activity (P<0.01) and a 68% inhibition in lipolysis (P<0.01), whereas diazoxide (10 microM) completely prevented each of these effects. These data demonstrate that human adipocytes express a SUR that regulates [Ca2+]i and, consequently, exerts coordinate control over lipogenesis and lipolysis. Accordingly, the adipocyte SUR1 may represent an important target for the development of therapeutic interventions in obesity.

[Effect of thyroid hormones on the modulation of genetic expression of liver cytosolic malic enzyme, in rats poisoned with hexachlorobenzene]. / Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of laboratory animals to HCB triggers porphyria, induction of liver microsomal enzymes, low levels of T4 reproductive dysfunction's, liver and thyroid tumors. Previous findings from our laboratory have shown that HCB increased the activity of the liver thyroid-responsive enzymes: malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) without any change in the mytochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD). In this study we have demonstrated that HCB treatment increased ME mRNA. We also have investigated if HCB affected: a) the thyroid hormone receptor (TR) concentration and binding affinity for its ligands, b) specifically the ME gene expression, or other thyroid hormone responsive enzymes were affected as well, c) Protein/DNA complex formed on the thyroid responsive element (TRE). Livers from female Wistar rats intoxicated with HCB (100 mg/100 g b.w.), for 9 and 15 days, were analyzed. Northern blot hybridization analysis, have demonstrated that ME mRNA levels increased 4 times and 2 times after 9 and 15 days intoxication respectively, without any alterations in the mRNA levels of other thyroid hormone responsive enzymes such as glyceraldheyde 3- phosphate dehydrogenase, phosphoenolpyruvatecarboxikinase and alpha-GPD. These results suggest that HCB affects specifically, ME gene expression. Hepatic T3 and T4 levels evaluated by RIA were not affected by HCB. Scatchard analyses showed that TR affinity and number of sites were not altered after 9 and 15 days of HCB treatment (control, Ka: 1.9 nM, Bmax 3.9 f/mol 100 micrograms DNA: HCD 9 days Ka: 2.1 nM, Bmax 4.5 fmol/100 micrograms DNA: HCB 15 days Ka 1.9 nM. Bmax 5.1 fmol/100 micrograms DNA intoxication, neither at 9 nor at 15 days. Electrophoresis mobility shift assay showed that HCB did not modify nuclear protein extract affinity for the TREs sequence. Our results suggest that TR itself was not directly involved in the induction of ME gene expression by HCB. Nevertheless TR could interact with other transcription factors in the overexpression of ME gene.

Rat mitochondrial glycerol-3-phosphate dehydrogenase gene: multiple promoters, high levels in brown adipose tissue, and tissue-specific regulation by thyroid hormone.

Mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (mtGPDH) is one of the two enzymes of the glycerol phosphate shuttle. This shuttle transfers reducing equivalents from the cytoplasm to the mitochondria in a unidirectional, exothermic manner. Here, the isolation and characterization of the rat nuclear gene (Gpd2) encoding mtGPDH is reported. The mtGPDH gene spans 100 kb and consists of 17 exons. The use of alternate promoters was suggested by the presence of three different first exons and confirmed by transient expression for two of them. The first exons are expressed in a tissue-restricted manner. Exon 1a was found primarily in brain, exon 1b was used in all tissues examined, and exon 1c was detected predominantly in testis. Depending on the tissue, different transcript lengths were also observed: 5.9 kb (all tissues), 3.6 kb (skeletal muscle), and 2.5 kb (testis). The length isoforms are attributable to alternate splicing and polyadenylation site use. Very high mtGPDH mRNA levels were found in brown adipose tissue, 75 fold greater than in white adipose tissue. Thyroid hormone increased mtGPDH mRNA levels in liver and heart but not in brown adipose tissue, brain, or testis. This pattern corresponds to that of thyroid hormone-induced oxygen consumption and is consistent with a role for mtGPDH in thyroid hormone-induced thermogenesis. Both thyroid-responsive and nonresponsive tissues used promoter 1b, suggesting that tissue-specific factor(s) contribute to the tissue-restricted responsiveness to thyroid hormone.

Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno / Action of the thyroid hormone on modulation of the gene expression of the liver cytosolic malic enzyme, in intoxicated rats with hexachlorobenzene

El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM.

Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno / Action of the thyroid hormone on modulation of the gene expression of the liver cytosolic malic enzyme, in intoxicated rats with hexachlorobenzene

El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM. (AU)

Physical performance and skeletal muscle characteristics after 2-week hind-limb unweighting.

It is well-known that 2 weeks of hind-limb suspension or space flight induce the sufficient decrease of the physical performance and simultaneously changes of muscle contractile properties and fiber size. However, the data on enzyme activities changes at present are contradictory. Numerous authors have pointed to the increase, reduction of its activity as well as its stability after experiments of the similar design. In previous studies it was shown that beta-GPA (beta-guanidino-propionic acid) administration increased the oxidative enzyme activities in the skeletal muscles and improved their contractile properties in hind-limb suspended rats. The aim of our study is to clear out what determines changes of the physical performance after 2 weeks of hind-limb suspension and beta-GPA administration.