The binding of trivalent chromium to low-molecular-weight chromium-binding substance (LMWCr) and the transfer of chromium from transferrin and chromium picolinate to LMWCr

A recent model for the role of chromium in insulin signaling requires that the oligopeptide low-molecular-weight chromium-binding substance (LMWCr) tightly bind four chromic ions before the oligopeptide obtains a conformation required for binding to the tyrosine kinase active site of the insulin receptor. To test this model, the chromium-binding constant of LMWCr was determined, and the ability of LMWCr to remove chromium from Cr2-transferrin and the nutritional supplement chromium picolinate, Cr(pic)3, was examined. These results are consistent with the model of the mode of action of LMWCr; a Hill study indicates the four chromic ions bind to apoLMWCr in a highly cooperative fashion (n =3.47) with a binding constant of 1.54x 10(21). Chromium is readily transferred from transferrin to apoLMWCr at near neutral pH. The results also suggest that reduction of the chromic center of Cr(pic)3 may be required for the supplement to release chromium; thus, release of chromium is related to a mechanism by which Cr(pic)3 may generate hydroxyl radicals in cells.     

Low-molecular-weight chromium-binding substance from chicken liver and American alligator liver

Low-molecular-weight chromium-binding substance (LMWCr), also known as chromodulin, is a chromium-binding oligopeptide proposed to have a function in chromium transport and insulin signaling in mammals. In this work, LMWCr has been isolated and purified for the first time from non-mammalian sources: chicken and American alligator. Milligram quantities of the oligopeptide can be obtained from kilogram quantities of liver. The LMWCr's from both sources are asparatate- and glutamate-rich oligopeptides which possess multinuclear chromium assemblies. The composition and physical and spectroscopic properties of the avian and reptilian LMWCr's are extremely similar to those of their mammalian analogues, suggesting the multinuclear sites of the biomolecule from all three classes of animal possess very similar structures. The chicken and alligator oligopeptides may possess intrinsic phosphotyrosine phosphatase activity.     

Spectroscopic and biological activity studies of the chromium-binding peptide EEEEGDD

While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr³⁺ to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.     

Evidence that chromium is an essential factor for biological activity of low-molecular-weight, chromium-binding substance

Biologically active Low-molecular-weight, chromium-binding substance (LMCr) has been isolated from the liver of rabbits injected with potassium bicarbonate and cow's milk. This substance enhances glucose oxidation and lipogenesis from glucose in rat adipocytes [Yamamoto A. et al. (1987) Eur. J. Biochem. 165, 627-631; (1988) J. Nut. 118, 39-45]. LMCr was shown to lose its activity almost completely as an effectant of glucose metabolism when Cr was deleted under acidic conditions and separated from LMCr by EDTA-chelation and successive molecular-sieve chromatography. Reincorporation of Cr3+ into apo-LMCr resulted in 50-90% recovery of its original activity. These findings suggest that the biological activity of LMCr resides in Cr.     

Isolation and Characterization of Low-Molecular-Weight Chromium-binding Substance (LMWCr) from Chicken Liver

Chromodulin (also known as low-molecular-weight chromium-binding substance, LMWCr) is a chromium-binding oligopeptide proposed to play a role in insulin signaling and chromium transport in mammals. This laboratory has isolated and purified this material from a non-mammalian source, an avian. Spectroscopic and physical characterization of the isolated material suggests the material is an oligopeptide with a multinuclear chromium assembly bridged via asparatate and glutamate residues very similar to its mammalian counterparts. The isolated material also possesses a biological activity similar to other LMWCr isolates.     

Metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol in alveolar macrophages from rat lung--II. Effects of intratracheal instillation of 3.4 mumol K2Cr2O7

Activity of the steroid 5 alpha-reductase in pulmonary alveolar macrophages from adult male rats has been investigated in vitro. Intratracheal instillation of 3.4 mumol K2Cr2O7 lowered the enzyme activity within 6 h, and the reduction was significant on the subsequent 2, 4 and 7 days. The activity of this enzyme was significantly decreased only 6 and 24 h after instillation when measured in the 800 g supernatant fraction of whole lung. Instillation of 3.4 mumol K2Cr2O7 increased serum levels of corticosterone. Serum levels of triiodothyronine and thyroxine decreased except for a transient increase 3 h after the K2Cr2O7 instillation. Subcutaneous administration of 200 micrograms dexamethasone/100 g b.wt, 200 micrograms/100 g b.wt of testosterone, 17 beta-hydroxy-5 alpha-androstane-3-one (5 alpha-DHT), dehydroepiandrosterone or corticosterone had no effect on the 5 alpha-reductase activity of the pulmonary alveolar macrophages within 12 h. The combined treatment with dexamethasone s.c. and intratracheal instillation of 3.4 mumol K2Cr2O7 reduced the steroid 5 alpha-reductase activity in the pulmonary alveolar macrophages to about 25% of controls. Measurement of the steroid 5 alpha-reductase activity in pulmonary alveolar macrophages as an index of lung damage when exposed to toxic material is discussed.     

The biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma cholesterol and triglycerides in rats: towards chromium-containing therapeutics

3 O(O₂CCH₂CH₃)₆ (H₂O)₃]⁺ 1 and a naturally occurring, biologically active form of chromium, low-molecular-weight chromium-binding substance (LMWCr), to rats are described. Given that the complexes are proposed to function by interacting with insulin receptor, trapping it in its active conformation, in contrast to current chromium-containing nutrition supplements, which only serve as sources of absorbable chromium, changes in lipid and carbohydrate metabolism would be expected. After 12 weeks administration (20 μg/kg body mass), compound 1 results in 40% lower levels of blood plasma LDL cholesterol, 33% lower levels of total cholesterol, and significantly lower HDL cholesterol and triglyceride; these results are in stark contrast to those of administration of other forms of Cr(III) to rats, which have no effect on these parameters. LMWCr, in contrast to 1, has no effect as it probably is degraded in vivoor excreted. These results are interpreted in terms of the mechanism of chromium action in response to insulin and the activation of insulin receptor, and the potential for the rational design of chromium-containing therapeutics is discussed. Received: 27 May 1999 / Accepted: 4 October 1999     

Isolation of a biologically active low-molecular-mass chromium compound from rabbit liver

A low-molecular-mass chromium-binding substance (LMCr), which is recognized as a detoxification ligand of chromium, was isolated from the livers of rabbits injected intravenously with K2Cr2O7 (200 mumol Cr/kg body wt) as a biologically active form. LMCr appears as an anionic, organic Cr compound with a relative molecular mass of 1500. It is composed of glutamic acid or glutamine, glycine, cysteine and aspartic acid or asparagine with a Cr/amino-terminal residue ratio of 4:1. The purified LMCr (10-300 ng Cr/ml) shows in vitro activities comparable to those of glucose tolerance factor in relation to insulin action. In the presence of insulin it enhances [U-14C]glucose conversion to 14CO (23-30% up) in rat epididymal adipocytes above the value obtained with insulin alone. LMCr also stimulates the rate of [3-3H]glucose incorporation into lipid by 30-40% with insulin or by 15-23% without insulin, as compared with the basic value obtained with insulin alone or without insulin. These findings suggest that LMCr plays essential roles in both glucose metabolism and detoxification of invaded Cr in the body.     

The trail of chromium(III) in vivo from the blood to the urine: the roles of transferrin and chromodulin

The chromium-binding oligopeptide chromodulin (also known as low-molecular-weight chromium-binding substance) has been shown to activate the tyrosine kinase activity of the insulin receptor in response to insulin and has been proposed to be part of a novel autoamplification mechanism for insulin signaling. The model requires that Cr3+ be moved from the blood to insulin-sensitive tissues in response to insulin and subsequently be lost in the urine as chromodulin; however, the model has not been tested by in vivo studies. In vivo studies with rats have shown that the iron transport protein transferrin serves as the major chromic ion transport agent and that this transport is stimulated by insulin. The ion is transported to a variety of tissues, while liver and kidneys are the major target. In hepatocytes, chromodulin occurs in appreciable levels in the cytosol and in the nucleus. Apochromodulin levels appear to be maintained under homeostatic control, although the only detectable form of urinary chromium is probably chromodulin. Increases in urinary chromium loss in response to insulin are reflected by increases in chromodulin, establishing a direct link between carbohydrate metabolism and the oligopeptide.     

Chromium in carbohydrate and lipid metabolism

 Since the discovery in the 1950s that mammals have a nutritional requirement for chromium, the biological function of chromium has been sought. Candidates for the naturally-occurring biologically active form of chromium have been proposed, but, until recently, all have been shown to be artifacts. Recent studies examining the properties of the oligopeptide low-molecular-weight chromium-binding substance (LMWCr) suggest that this material may have a role in carbohydrate and lipid metabolism as part of a novel insulin-signaling amplification mechanism and may have implications in the treatment of diabetes and related conditions. Received: 24 March 1997 / Accepted: 9 September 1997     

Corticosteroids and lung surfactant levels in adult male rats

Following lung instillation in adult male rats of 3.4 mumol hexavalent chromium (K2Cr2O7) dissolved in 0.5 ml of 0.9% NaCl, increased levels of lung surfactant could be detected after 48 h. The blood serum concentration of corticosterone was elevated in these animals. Blood serum thyroxine and triiodothyronine showed an initial increase after lung instillation of hexavalent chromium followed by a decline. Metabolism of testosterone by the alveolar macrophages to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol was reduced 6 and 12 h after the K2Cr2O7 instillation, which was also associated with damage of lung cell function and decreased uptake by the alveolar macrophages of Candida albicans particles. As early as 12 h after s.c. administration of 400 micrograms dexamethasone/100 g body wt, increased levels of lung surfactant could be measured. At this time the lungs showed no signs of cellular damage, and metabolism of testosterone as well as uptake of Candida albicans particles by the alveolar macrophages were normal. Lower s.c. doses of dexamethasone did not result in raising the levels of lung surfactant in 12 h. Within 12 h after s.c. administration of large doses of testosterone, dihydrotestosterone or dehydroepiandrosterone no measurable effects on the levels of lung surfactant could be measured. Since animals treated with dexamethasone (200 micrograms/100 g body wt) or long-acting synthetic ACTH (100 micrograms i.m. Synacthen Depot/100 g body wt) for 5 days after lung instillation of K2Cr2O7 had extremely high levels of lung surfactant, it is concluded that the corticosteroids in adult rats may help to create augmented surfactant levels following lung intoxication. This could proceed via stimulation of surfactant production and reduction of surfactant removal. Different aspects of lung surfactant metabolism are discussed.     

Regulation of urea synthesis in sublobular regions of the liver lobule by oxygen

Periportal and pericentral regions of the liver lobule were isolated from perfused rat liver using a micropunch and incubated in Krebs-Henseleit buffer (pH 7.6) containing 2% poly(ethylene glycol) in Eagle's basal medium, PMSF (50 micrograms/ml) and leupeptin (20 micrograms/ml) for 2 h at 25 degrees C under and O2/CO2 (95:5%) gas phase. Maximal rates of urea production from ammonium chloride were 96.4 +/- 8.7 and 32.8 +/- 5.4 mumol/g per h at 800 and 200 microM O2. Thus, urea synthesis was 2-3-times greater at high than low O2 tension in plugs from periportal and pericentral regions of the liver lobule.     

Concentrations of essential elements after repeated administrations of tin and selenium

To study effects of simultaneous administration of tin (Sn) and selenium (Se) on concentrations of several essential elements, mice were injected with either SnCl2 (ip) or Na2SeO3 (sc), alone or both compounds at a daily dose of 5 mumol/kg each for 12 consecutive days. Mice were sacrificed at 20 h after the last injection and concentrations of Sn, Se, Na, Ca, Zn, P, Fe, K, and Mg in the liver, kidney, spleen, pancreas, testis, seminal vesicle, lung, femoral muscle, and femoral bone were determined. In the control mice, Sn and Se concentrations were the highest in bone (0.69 micrograms Sn and 6.93 micrograms Se/g dry wt). Administered Sn was found to accumulate in all organs except the testis. Among the essential elements determined, Na was the most affected in terms of concentration in the organs and Mg was the least affected element in these organs. Among the organs tested, each elemental concentration in the pancreas was most affected. Simultaneous injections of Sn and Se appeared to keep the correlation coefficients between elements similar to those found in the control mice.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

Study of liver differentiation in vitro

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.     

Thyroid hormone effects upon Na+K+ dependent adenosine triphosphatase activity of microsomal fraction of liver, muscle and brain of toad Bufo melanostictus

Na+K(+)-ATPase activity in the liver and muscle microsomal membranes have been determined by different doses (0.1, 0.25, 0.5, 1 and 2 micrograms/gm of body weight) of L-triiodothyronine and L-thyroxine in the toad, Bufo melanostictus. The minimum effective dose of T3 was 0.5 microgram/g in case of both liver and muscle to stimulate the enzyme activity and there was dose dependent rise between T3 at the doses of 0.5 and 1 microgram/g. T3 at the doses of 1 and 2 micrograms/g produced more or less the same level of activity. T4 showed an increased activity at 1 and 2 micrograms/g without any dose dependent fashion in the two organs. The doses 0.1 and 0.25 microgram/gm body weight of T3 and 0.1, 0.25 and 0.5 microgram/gm body weight of T4 remained ineffective to elicit any response in both organs. The grain showed no significant change in the enzyme activity at any of the applied doses of T3 and T4. Cycloheximide inhibited T3 induced rise in Na+K(+)-ATPase activity of liver and muscle. Treatment with propylthiouracil caused a significant fall in Na+K(+)-ATPase activity of liver and muscle and the normal value was restored in the two organs after three consecutive injections of T4 at the dose of 1 microgram/g.     

Enzyme cytochemistry combined with electron microscopy, pharmacokinetics, and clinical chemistry for the evaluation of the effects of steady-state valproic acid concentrations on the mouse

A number of organs from adult female mice were investigated after continuous application of the anticonvulsant drug valproic acid (VPA) by enzyme cytochemistry, light and electron microscopy, pharmacokinetics and clinical chemistry. VPA plasma levels were maintained between 55 micrograms/ml and 67 micrograms/ml for three days following subcutaneous implantation of drug reservoirs. Effects detectable by enzyme cytochemical or electron microscopical means were mainly observed in liver, kidney, thymus and spleen. A strict concentration-dependency of drug effects could not be found. In the liver, the activities of some surface-membrane hydrolases were increased at the biliary pole; the activities of other hydrolases were decreased or unchanged. Electron microscopically, number and length of microvilli of hepatocytes were increased and many of them showed fat inclusions, mitochondrial swellings and autophagic vacuoles. In some of the proximal convoluted tubules of the kidney, the reaction product originating from microvillous and lysosomal hydrolases was diffusely distributed and its amount lowered. This was paralleled by tubular cells with an increased number of fat droplets and swollen mitochondria or destroyed tubular cells, as demonstrated by electron microscopy. Additionally, peritubular endothelial cells were arranged in a garland-like pattern. Alkaline phosphatase was activated in the straight portion of the proximal tubules. Increased glucose, creatinine and total protein concentrations and increased gamma-glutamyl transpeptidase and alkaline phosphatase activities in the urine reflected well the damage of the proximal renal tubules. Cortical and medullary morphology varied considerably in the thymus. In extreme cases, the cortical zone was either reduced in size or the medulla showed a cortex-like structure or vice versa (inverted type of thymus). The thymic cortical reticular cells showed increased aminopeptidase A activity accompanied by a generalized aminopeptidase M and alkaline phosphatase reaction. Our data indicate that--in addition to the liver--also the kidney, thymus and spleen are target organs of VPA-induced toxicity in the mouse.     

Binding of manganese in human and rat plasma

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (109 Cd2+, 65Zn2+, 59Fe3+), 54Mn2+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 65Zn-albumin in the presence of excess Mn2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as Fe3+, but better than Zn2+ or Cd2+. Saturation of transferrin with Fe3+ (1.6 micrograms Fe/mg) prevented the binding of 54Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin rather than with albumin in both human and rat plasma. The amount of 54Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn2+ to Mn3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of 54Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include alpha 2-macroglobulin, heavy gamma-globulins and/or heavy lipoproteins.     

Albumin and transferrin synthesis during development in the rat

In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.     

Effect of chronic treatment with neuroleptics on the content of 3',5'-cyclic guanosine monophosphate in cerebellar cortex of rats.

Chronic treatment with haloperidol is associated with complete tolerance to the decreasing effect of the neuroleptic on cerebellar cGMP content, vice versa chronic haloperidol causes hypersensitivity to the enhancing effect of apomorphine on cerebellar cGMP. Thus, the administration of 0.5 mg/Kg of haloperidol decreases cerebellar cGMP by 80% in control rats but fails to alter this nucleotide in rats chronically treated with haloperidol (0.5 mg/Kg twice daily for 20 days). A dose of 0.5 mg/Kg of apomorphine enhances cGMP by approximately 25 and 60 percent in control rats and in rats chronically treated with haloperidol, respectively. The results suggest that: a) There is a functional link between striatum and cerebellum; b) Cerebellar cGMP is a sensitive index of the state of activation of striatal dopamine receptors.