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.

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.

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.

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.

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.

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.

Effect of antispermatogenic compound CDRI-84/35 on marker enzymes of rat testis cells. A study on site of action.

Marker enzymes of Sertoli and germ cells were estimated to study the mechanism of action of antispermatogenic compound CDRI 84/35 in adult male rat testis. Animals were killed after 22, 41, and 64 days of treatment with antispermatogenic dose of CDRI 84/35 in order to evaluate the effect of the compound on spermatid, spermatocyte, and spermatogonial stages, respectively. Studies were also extended to a recovery period of 90 days. Results indicate a direction action of the compound on germ cells, with no apparent effect on Sertoli cells. Studies also show a massive depletion of postmeiotic germ cells after the treatment, with some damage to premeiotic germ cells as well. Reversibility of the compound was partial, with the marker enzymes of pre- and postmeiotic germ cells not being restored to control levels after withdrawal of treatment.

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.

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.

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.

[The effect of taurine on the activity of transport ATPases and of energy metabolism enzymes in different tissues of rats with acute hypoxic hypoxia]. / Vliianie taurina na aktivnost' transportnykh ATP-az i fermentov énergeticheskogo obmena v raznykh tkaniakh krys pri ostroi gipoksicheskoi gipoksii.

The membrane activity of Na+, K(+)-ATPase, Mg2+, Ca(2+)-ATPase, mitochondrial NAD-isocitrate dehydrogenase, mitochondrial and cytosolic L-glycerol-3-phosphate dehydrogenase was determined in the liver and brain of Wistar rats under acute hypoxic hypoxia against the background of preventive taurine administration. It was shown that preliminary taurine treatment prevented a decrease of hypoxia in activity of Na+. K(+)-ATPase and mitochondrial calcium-dependent enzymes, mostly in the liver. Changes in the intracellular calcium content and biomembrane structure have been discussed as the mechanisms of the taurine effect on the enzymes' activity.

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.

Effects of dehydroepiandrosterone in rats injected with streptozotocin during the neonatal period.

Control rats and diabetic animals injected with streptozotocin during the neonatal period were either maintained on a standard diet or given access to food supplemented with dehydroepiandrosterone (DHEA, 0.2%) for 11 days before sacrifice. In both control and diabetic rats, DHEA feeding augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in liver, but not so in either the parotid gland or pancreatic islets. DHEA lowered, in both control and diabetic rats, the ratio between D-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration of D-glucose, as well as the insulin concentration and insulin/glucose ratio in plasma. These findings support the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the otherwise unfavorable consequences of chronic hyperactivity.

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.

Streptozotocin-induced suppression of FAD-linked glycerophosphate dehydrogenase in pancreatic islets of adult rats.

Injection of streptozotocin (30-40 mg/kg body weight) to adult rats caused within 4-6 days a sizeable decrease in the activity of FAD-linked glycerophosphate dehydrogenase in pancreatic islets, with little change in either glutamate dehydrogenase or 2-oxoglutarate dehydrogenase activity. The severity of the enzymatic defect was related to that of the diabetic state, although a decreased enzymic activity was also observed in islets from virtually normoglycemic animals examined 2-3 weeks after streptozotocin injection. The administration of nicotinamide prior to that of streptozotocin prevented the change in enzymic activity. It is proposed that the enzymatic defect, rather than being attributable to a genomic effect of streptozotocin, may reflect the preferential impairment of a subpopulation of pancreatic B-cells.

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.

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.

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.