Naringin prevents bone loss in a rat model of type 1 Diabetes mellitus.

The aim of this work was to know whether naringin (NA) could prevent the bone complications in a model of streptozotocin (STZ) induced diabetes. Rats were divided in: 1) controls, 2) STZ-rats, 3) STZ-rats treated with 40 mg NA/kg, and 4) STZ-rats treated with 80 mg NA/kg. BMD and BMC were performed by DEXA. Bone histomorphometry and histology as well as TRAP staining were done in tibia. Osteocalcin (OCN) was determined in bone and serum. Glutathione content and SOD and catalase activities were assayed in bone marrow from femur. The data showed that NA80 increased the BMD and BMC from the long bones of STZ-rats. Both NA40 and NA80 normalized the trabecular number and the trabecular separations. An increase in the number of adipocytes and TRAP(+) cells in tibia from STZ-rats was blocked by NA. NA40 treatment increased the number of OCN(+) cells, but only the NA80 treatment allowed to reach the control values. NA normalized the SOD and catalase activities in bone marrow of femur from STZ-rats. In conclusion, NA avoids alterations in the physical properties and microstructure of bone from STZ-rats probably by stimulation of osteoblastogenesis, inhibition of the osteoclastogenesis and adipogenesis via blocking the oxidative stress.

Naringin prevents the inhibition of intestinal Ca2+ absorption induced by a fructose rich diet.

This study tries to elucidate the mechanisms by which fructose rich diets (FRD) inhibit the rat intestinal Ca2+ absorption, and determine if any or all underlying alterations are prevented by naringin (NAR). Male rats were divided into: 1) controls, 2) treated with FRD, 3) treated with FRD and NAR. The intestinal Ca2+ absorption and proteins of the transcellular and paracellular Ca2+ pathways were measured. Oxidative/nitrosative stress and inflammation parameters were evaluated. FRD rats showed inhibition of the intestinal Ca2+ absorption and decrease in the protein expression of molecules of both Ca2+ pathways, which were blocked by NAR. FRD rats showed an increase in the superoxide anion, a decrease in the glutathione and in the enzymatic activities of the antioxidant system, as well as an increase in the NO content and in the nitrotyrosine content of proteins. They also exhibited an increase in both IL-6 and nuclear NF-κB. All these changes were prevented by NAR. In conclusion, FRD inhibit both pathways of the intestinal Ca2+ absorption due to the oxidative/nitrosative stress and inflammation. Since NAR prevents the oxidative/nitrosative stress and inflammation, it might be a drug to avoid alteration in the intestinal Ca2+ absorption caused by FRD.

Antioxidant and antiapoptotic properties of melatonin restore intestinal calcium absorption altered by menadione.

The intestinal Ca²âº absorption is inhibited by menadione (MEN) through oxidative stress and apoptosis. The aim of this study was to elucidate whether the antioxidant and antiapoptotic properties of melatonin (MEL) could protect the gut against the oxidant MEN. For this purpose, 4-week-old chicks were divided into four groups: (1) controls, (2) treated i.p. with MEN (2.5 µmol/kg of b.w.), (3) treated i.p. with MEL (10 mg/kg of b.w.), and (4) treated with 10 mg MEL/kg of b.w after 2.5 µmol MEN/kg of b.w. Oxidative stress was assessed by determination of glutathione (GSH) and protein carbonyl contents as well as antioxidant enzyme activities. Apoptosis was assayed by the TUNEL technique, protein expression, and activity of caspase 3. The data show that MEL restores the intestinal Ca²âº absorption altered by MEN. In addition, MEL reversed the effects caused by MEN such as decrease in GSH levels, increase in the carbonyl content, alteration in mitochondrial membrane permeability, and enhancement of superoxide dismutase and catalase activities. Apoptosis triggered by MEN in the intestinal cells was arrested by MEL, as indicated by normalization of the mitochondrial membrane permeability, caspase 3 activity, and DNA fragmentation. In conclusion, MEL reverses the inhibition of intestinal Ca²âº absorption produced by MEN counteracting oxidative stress and apoptosis. These findings suggest that MEL could be a potential drug of choice for the reversal of impaired intestinal Ca²âº absorption in certain gut disorders that occur with oxidative stress and apoptosis.

New perspectives in melatonin uses.

This review summarizes the metabolism, secretion, regulation and sites of action of melatonin. An updated description of the melatonin receptors, including their signal transduction mechanisms, distribution and characterization of receptor genes, is given. Special emphasis is focused on the clinical aspects and potential uses of melatonin in the sleep-wake rhythms, in the immune function, in cancer therapy, in neuroprotection against oxidative damage and antioxidant activities in different tissues. Finally, combined effects of melatonin with other drugs are discussed.

[Metabolic alterations triggered by low calcium diets]. / La restricción dietaria de calcio produce alteraciones metabólicas.

Calcium is essential for bone and tooth formation, achievement of optimal peak bone mass and also for regulation of physiological processes. The calcium demand depends on age, gender and different physiological processes. These requirements are higher during childhood, pregnancy and lactation. Dietary Ca2+ deficiency modifies Ca2+ homeostasis and the metabolism of calciotropic hormones and increases the efficiency of intestinal Ca2+ absorption and renal reabsorption, altering bone metabolism. The low Ca2+ diet is associated with hypertension and risk of cancer.

Effect of retinal ablation on the expression of calbindin D28k and GABA in the chick optic tectum.

The effect of retinal ablation on qualitative and quantitative changes of calbindin D28k and GABA expression in the contralateral optic tectum was studied in young chicks. Fifteen days old chicks had unilateral retinal ablation and after 7 or 15 days, calbindin expression was analyzed by Western blot and immunocytochemistry. Neuronal degeneration was followed by the amino-cupric silver technique. After 15 days, retinal lesions produced a significant decrease in calbindin immunostaining in the neuropil of layers 5-6 and in the somata of neurons from the layers 8 and 10 of the contralateral tectum, being this effect less marked at 7 days post-lesion. Double staining revealed that 50-60% of cells in the layers 8 and 10 were calbindin and GABA positive, 30-45% were only calbindin positive and 5-10% were only GABAergic neurons. Retinal ablation also produced a decrease in the GABA expression at either 7 or 15 days after surgery. At 7 days, dense silver staining was observed in the layers 5-6 from the optic tectum contralateral to the retinal ablation, which mainly represented neuropil that would come from processes of retinal ganglion cells. Tectal neuronal bodies were not stained with silver, although some neurons were surrounded by coarse granular silver deposits. In conclusion, most of calbindin molecules are present in neurons of the tectal GABAergic inhibitory circuitry, whose functioning apparently depends on the integrity of the visual input. A possible role of calbindin in the control of intracellular Ca2+ in neurons of this circuit when the visual transmission arrives to the optic tectum remains to be studied.

Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development.

The immunolocalization of the plasma membrane calcium pump (PMCA) was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP) immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody SF10 from human erythrocyte plasma membrane cross-reacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.

DL-Buthionine-S,R-sulfoximine affects intestinal alkaline phosphatase activity.

The susceptibility of intestinal alkaline phosphatase to DL-buthionine-S,R-sulfoximine was investigated in chicks fed a commercial diet. The results show that DL-buthionine-S,R-sulfoximine produced inhibition of intestinal alkaline phosphatase activity. This effect showed dose- and time-dependency and it was caused by either in vivo DL-buthionine-S,R- sulfoximine administration or in vitro DL-buthionine-S,R-sulfoximine incubation with villus tip enterocytes. DL-Buthionine-S,R-sulfoximine did not act directly on intestinal alkaline phosphatase but it provoked glutathione depletion which led to changes in the redox state of the enterocyte as shown by the production of free hydroxyl radicals and an incremental increase in the carbonyl content of proteins. The reversibility of the buthionine sulfoximine effect on intestinal alkaline phosphatase was proved by addition of glutathione monoester to the duodenal loop.

Effect of vitamin D deficiency on lipid composition and calcium transport in basolateral membrane vesicles from chick intestine.

Vitamin D deficiency affects the lipid composition and Ca2+ uptake of intestinal basolateral membranes from chick intestine. The increased cholesterol content causes an increase in the molar ratio cholesterol/phospholipid. Phospholipid classes remain unchanged, but the percentages of arachidonic acid from the from the major phospholipid fractions are increased. After 24 hours of oral administration of 2,000 IU of cholecalciferol to vitamin D-deficient chicks, the cholesterol values do not change, but the amount of arachidonic acid returns to normal values. Ca2+ uptake driven by ATP is diminished in vesicles from intestinal basolateral membranes of vitamin D-deficient chicks. Cholecalciferol treatment returns these values to the controls which might be due mainly to the increased number of Ca2+ pump units. In conclusion, changes in lipid composition and in Ca2+ pump caused by vitamin D deficiency seems to play a role in the decrease of vesicular Ca2+ transport. A single dose of cholecalciferol restores only partially the lipid-protein changes produced by vitamin D deficiency.

Calcium and phosphorous deficiencies alter the lipid composition and fluidity of intestinal basolateral membranes.

Steady-state fluorescence polarization and lipid composition studies were undertaken on intestinal basolateral membranes (BLM) from chicks adapted to a calcium deficient (low Ca) or a phosphorous deficient diet (low P). The fluorescence anisotropy showed that fluidity of intestinal BLM was increased by the mineral deprivations, but the response of the membranes varied with the specific fluorophore used. The "static" component of fluidity, assessed by 1,6-diphenyl-1,3,5-hexatriene (DPH), was increased whereas the "dynamic" component, monitored with DL-12-(9-anthroyloxy)-stearic acid (12-AS), was not modified. Low P diet produced significant changes in lipid composition such as a decrease in the cholesterol content and in the sphyngomyelin (Sph) and phosphatidylserine plus phosphatidylinositol fractions (PS + PI) and increment in the phosphatidylcholine (PC) proportion. The percent of monounsaturated fatty acids was increased by the low P diet due mainly to an increase in the oleic acid fraction. Minor changes such as a decrease in the palmitic acid and increases in the 22:5n3 and 22:6n3 fatty acids were caused by Ca deficiency. The alteration of the biochemical and biophysical membrane properties of the BLM of the mineral deficient groups might play a role in the enhanced intestinal Ca and P absorption.

Glutathione plays a role in the chick intestinal calcium absorption.

DL-buthionine-S,R-sulfoximine (BSO) administration to vitamin D-deficient chicks treated with cholecalciferol produces a rapid decrease in the Ca2+ transfer from lumen-to-plasma and in the intestinal glutathione content. This response was reversed by addition of glutathione monoester to the intestinal sac. Variables related to the Ca2+ homeostasis such as plasma Ca and P, and intestinal calbindin D28k were not modified by BSO given to vitamin D-deficient chicks treated with cholecalciferol. Intestinal alkaline phosphatase activity, on the contrary, was highly reduced by BSO in vitamin D-deficient chicks treated with vitamin D3. This effect showed time and dose-dependency. Although the mechanism/s of action of BSO on the intestinal Ca absorption is unknown, it is quite possible that thiol groups of protein involved in the Ca2+ transport are affected by the GSH depletion and/or by block of the antioxidant ability of vitamin D3. Thus, reactive oxygen compounds would be increased and, therefore, the Ca2+ movement from lumen to plasma decreases.

Vitamin D affects Krebs cycle NAD-linked oxidoreductases from chick intestinal mucosa.

Vitamin D3 administration affects the NAD-linked oxidoreductase activities of Krebs cycle from intestinal mucosa of vitamin D-deficient chicks. Vmax values were increased in all of them, while K0.5 for substrate remained unchanged except for 2-oxoglutarate dehydrogenase, which showed lower affinity for oxoglutarate. Addition of Ca2+ to the incubation medium increased the affinity of 2-oxoglutarate dehydrogenase and NAD-isocitrate dehydrogenase for their substrates either in the vitamin D3 treated group or in the control one. The activity of succinate dehydrogenase, a FMN-dependent oxidoreductase, was not modified by vitamin D3 administration. The oxygen consumption of the intestinal mitochondria was not altered by cholecalciferol treatment to vitamin D-deficient chicks. The reason why vitamin D3 selectively affects the NAD-linked oxidoreductase activities of the Krebs cycle remains unknown. The vitamin D hormone, 1,25(OH)2D3, appears to be the mediator of the response.

Immunocytochemical localization of the plasma membrane calcium pump, calbindin-D28k, and parvalbumin in Purkinje cells of avian and mammalian cerebellum.

A monoclonal antibody produced against the human erythrocyte plasma membrane calcium pump (PMCA) was shown to react immunohistochemically with an epitope of the PMCA in avian and mammalian cerebellum. Western blot analysis of purified synaptosomes and homogenates from avian cerebellum revealed major immunoreactive proteins with molecular masses (130 kDa and 138 kDa) similar to those of purified erythrocyte PMCA. Dual-imaging confocal immunofluorescence microscopy of avian cerebellum showed that the PMCA antibody stained the periphery of the soma whereas calbindin-D28k was located in the cytosol. PMCA heavily stained the more distal dendrites of the Purkinje cells and, within the resolution of the fluorescence procedure, colocalized with calbindin-D28k. By using alkaline phosphatase-conjugated second antibody, PMCA was again localized to the peripheral soma, to a segmental pattern in dendrites, and to presumed spiny elements. The soma periphery and dendrites of Purkinje cells of the rat cerebellum were also prominently stained with anti-PMCA antibody and compared to parvalbumin localization. Dendritic depolarization and dendritic spiking behavior are significant Ca(2+)-dependent events of Purkinje cells. The rapid decline of intracellular free Ca2+ after the rapid rise time of Ca2+ transients is considered to be due to sequestration by Ca2+ buffers, uptake by intracellular stores, and Ca2+ extrusion mechanisms, the latter a function of PMCA now shown immunohistochemically to be a prominent feature of Purkinje cell dendrites.

Hormonal effects on the sulfhydryl groups associated with intestinal brush border membrane proteins.

Previous studies demonstrated that the administration of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) to cholecalciferol-deficient chicks rapidly increases the reactivity and amount of the sulfhydryl (HS-) groups in intestinal brush border membranes (BBM). In the present study, the tissue and hormonal specificity of this effect was investigated. The HS- groups of intestinal and renal BBM were enhanced by vitamin D-3 and/or 1,25(OH)2D3, but no change was noted in isolated intestinal mitochondria and purified intestinal basolateral membranes, cardiac sarcolemma and erythrocyte membranes. Other steroid hormones including estradiol, testosterone, aldosterone, cortisol, dexamethasone and progesterone, yielded a response similar to 1,25(OH)2D3 on BBM HS- groups. Triiodothyronine and retinoic acid also resulted in an increase in intestinal BBM HS- groups. In a kinetic approach, using a specific sulfhydryl fluorescent probe (N-7-dimethylamino-4-coumarin-3-yl-maleimide, DACM), the reactivity of the BBM HS- groups was increased by estrogen and testosterone, as was previously shown for 1,25(OH)2D3. Intestinal BBM proteins, labeled with DACM, were separated by gel electrophoresis. Fluorescence scans of the gel showed two heavily labeled bands, one of 110 kDa, putatively brush border myosin I, and one of 43 kDa, putatively actin. Labeling of the 110 kDa protein was increased by 1,25(OH)2D3 and estradiol. Further studies are required to elucidate the physiological meaning of these hormone-mediated increases in reactivity and amount of the BBM sulfhydryl groups, as well as the nature of the intermediate biochemical reactions involved in this response.

Enhancement of sulfhydryl group availability in the intestinal brush border membrane by deficiencies of dietary calcium and phosphorus in chicks.

The reactivity and availability of sulfhydryl (-SH) groups in brush border membranes (BBM) from chicks adapted to a calcium-deficient (low Ca) or a phosphorus-deficient (low P) diet were determined. The calbindin-D28K concentrations of the intestinal mucosa of the low Ca and low P groups were both increased approximately 2.5-fold, demonstrating that adaptation to the mineral deficiencies had occurred. By the Ellman reaction, a threefold increment in -SH groups in BBM from both mineral-deficient groups was noted. By using DACM (N-7-dimethylamino-4-methylcoumarin-3-yl maleimide), a fluorescent probe for -SH groups, it was observed that fluorescence development was considerably greater with BBM from the low Ca and low P groups than with BBM from the controls, whether measured in the absence or presence of sodium dodecyl sulfate (SDS). In the absence of SDS, the pseudo-first-order reaction rate constants, k', calculated from the fluorescence data, were greater than the control group values, but in the presence of SDS, the k' values for all groups were about the same. Similar changes in BBM-SH groups were previously observed when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient chicks. The redox state of the sulfhydryl groups in enzymes and transport proteins is known to affect the level of their activity. The functional significance of the present observations concerning the -SH groups of chick intestinal BBM, particularly in relation to vitamin D-dependent calcium and phosphorus absorption, is not known but is under investigation.

Vitamin D3 administration increases the membrane fluidity of intestinal mitochondria.

The fluorescence anisotropy in the mitochondria from vitamin D-treated chicks is significantly lower than that from the vitamin D-deficient animals with the inner core probe DPH. Surface membrane fluidity, measured with the probe TMA-DPH, shows no differences between the organelles of both groups. The fluorescence studies performed in mitochondrial subfractions revealed that cholecalciferol treatment induces a decrease of lipid order parameter S (DPH) in the mitochondrial inner membrane. These results pose the question of whether vitamin D3 participates in the regulation of physiological function of the intestinal mitochondria through changes in the physical properties of the membranes.

Phospholipid and fatty acid changes produced by cholecalciferol on intestinal mitochondrial membranes.

Cholecalciferol administration to vitamin D-deficient chicks produces, 24 h after treatment, a specific increase of the phosphatidylcholine content in the intestinal mitochondrial inner membrane plus matrix fraction without changes in its proportion in the outer membrane. The ratio of unsaturated/saturated fatty acids in the outer membrane phosphatidylcholine was increased by that treatment. The inner membrane plus matrix presents a decrease of 16:1 in phosphatidylethanolamine and 18:0 in the phosphatidylcholine fraction. Cardiolipin shows the largest change in the ratio of unsaturated/saturated fatty acids predominantly by an increase in the linoleic acid. The present data suggest that phosphatidylcholine and fatty acids modifications in both mitochondrial subfractions caused by vitamin D3 might have some role in the intestinal mitochondrial Ca transport.

Phospholipids and sialic acid changes produced by vitamin D3 on intestinal mitochondria.

Vitamin D3 administration to vitamin D-deficient chicks produces, 24 hours after treatment, a large increment of phospholipids in mitochondria from intestinal mucosa. Proportion of the different phospholipid classes and fatty acid composition of the organelles were not modified by that treatment. A time course study of the effects of 1,25(OH)2D3 on calcium absorption and sialic acid content of intestinal mitochondria glycoproteins showed that both effects were correlated. The results suggest that either vitamin D or the increase of calcium transfer are involved in the make up of intestinal mitochondria membranes.

Phosphate intestinal secretion and absorption by isolated ileal loop: effects of cholecalciferol and diet phosphate.

The effect of diet phosphate content and cholecalciferol on intestinal phosphate secretion and absorption was investigated in rachitic chicks. Phosphate absorption was determined by the in situ ligated loop technique. Phosphate secretion was estimated by a method proposed by the authors. Hydroxyapatite, placed in the lumen of a ligated loop, acts as trapping agent of 32P leaving the intestinal tissue. The fraction of rapid exchangeability of tissue phosphate was taken as the precursor pool of secreted phosphate. Control chicks fed diets containing 0.3% P (group 1) or 1.0% (group 2) showed similar Pi absorption; the secretion was larger for group 2. After cholecalciferol treatment for 2 or 4 consecutive days an increment of Pi absorption with simultaneous reduction of Pi secretion was evident for both groups of animals. Chicks treated for 7 days gave values similar to those of controls. It is concluded that the regulation of intestinal phosphate absorption and secretion could be one important mean of homeostatic control. Intestinal phosphate movement is adapted to dietary phosphate and is partially independent of cholecalciferol.