Nutritional aspects of manganese from experimental studies.

In experimental animals, dietary manganese deficiency can result in numerous biochemical and structural abnormalities. Deficient animals can be characterized by impaired insulin production, alterations in lipoprotein metabolism, an impaired oxidant defense system, and perturbations in growth factor metabolism. If the deficiency occurs during early development, there can be pronounced skeletal abnormalities and an irreversible ataxia. Several lines of evidence suggest that manganese deficiency may be a problem in some human populations. Manganese toxicity can also pose a significant health risk. In experimental animals, acute manganese toxicity can result in numerous biochemical pathologies. However, the above occurs typically when the manganese is given via injection; most animals show considerable resistance to dietary manganese toxicosis. Similarly, confirmed cases of manganese toxicity in humans are currently restricted to cases of exposure to high levels of airborne manganese, and to cases when manganese excretory pathways are compromised.

A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma.

A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure endogenous levels of vascular endothelial growth factor (VEGF165) in human plasma. The ELISA can be performed in 10% human EDTA plasma, yielding a neat plasma sensitivity of 10 pg/ml or 0.2 pM. The recovery of recombinant human VEGF (rhVEGF) added to human plasma ranges from 89 to 100%. The capture antibody depletes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coefficients of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18%, respectively. Characterization of the ELISA using plasmin derived VEGF variants suggests the assay is specific for the VEGF165 isoform. The heterodimer, VEGF(165/110) quantitates similar to that of the intact VEGF165 homodimer, however, the homodimers VEGF121, VEGF110 and the carboxy terminal domain (residues 111-165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal healthy individuals range from 20 to 141 pg/ml, with a mean of 42 +/- 22 pg/ml. There were no significant differences in VEGF levels between males and females. Circulating endogenous VEGF levels in cancer patients ranged from 32 to 418 pg/ml, averaging 129 +/- 17 pg/ml.

A His19 to Ala mutant of melanoma growth-stimulating activity is a partial antagonist of the CXCR2 receptor.

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.

The influence of manganese deficiency on serum IGF-1 and IGF binding proteins in the male rat.

Young male rats subjected to a dietary manganese (Mn) deficiency respond to the deficiency by reducing their growth rate. The growth hormone (GH)/insulin-like growth factor (IGF) axis is critical for linear growth; this system is exquisitely sensitive to the nutritional state of the animal. In this study, we examined circulating GH, IGF-1, and insulin levels in Mn-deficient (-Mn; fed a 0.5 microg Mn/g diet) and sufficient (+Mn; fed a 45 microg Mn/g diet) male Sprague-Dawley rats. Additionally, we examined the distribution of circulating IGF binding proteins (IGFBPs) in animals of both dietary groups as these proteins modulate IGF-1 action in vivo and in vitro, and have been demonstrated to be altered in a number of nutritional and physiological states. Body weight was significantly reduced in -Mn relative to +Mn rats. Consistent with other studies, daily food intake was not altered. However, cumulative food intake (over 3 months) was marginally lower in -Mn versus +Mn animals. -Mn animals displayed lower circulating concentrations of IGF-1 (66% of control levels) and insulin (60% of control levels) despite having significant elevations in circulating GH levels relative to +Mn animals (140% of control levels). The IGFBP profile of -Mn animals reflected their elevated GH status, as we observed increased binding of tracer (125I-IGF-1) to the circulating IGFBP-3 complex (120% of control binding) using native chromatography techniques. Interestingly, the lower circulating insulin concentrations of -Mn animals did not result in dramatic elevations in lower-molecular-weight binding proteins. In summary, we demonstrate that in young male rats, Mn deficiency is associated with alterations in IGF metabolism. These alterations may contribute to the growth and bone abnormalities observed in -Mn animals.

Effect of chronic IL-1 beta infusion on glucose homeostasis and pancreatic insulin secretion.

The present studies examined the effects of chronic interleukin-1 (IL-1 beta) infusion on glucose homeostasis and insulin secretion in male Sprague Dawley rats. IL-1 beta (4 micrograms per day) or saline was infused over a six day period using mini-osmotic pumps, surgically inserted under light ether anesthesia. Saline-infused rats were fed the amount of food consumed by their respective pair in the IL-1 beta group on the previous day. IL-1 beta infusion resulted in decreased food intake and significant body weight loss as well as increased liver and kidney weights. IL-1 beta infusion resulted in fasting hypoglycemia as well as elevated blood glucose levels in response to an oral glucose load compared to controls. Glucose-induced insulin secretion from the isolated perfused pancreas was significantly lower in IL-1 beta treated rats compared to controls. These data demonstrate that chronic IL-1 beta administration alters glucose homeostasis and impairs glucose-induced insulin secretion.

Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum.

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.

Pharmacokinetic profile of recombinant human (rh) inhibin A and activin A in the immature rat. I. Serum profile of rh-inhibin A and rh-activin A in the immature female rat.

The serum pharmacokinetics of recombinant human inhibin A (rh-inhibin A) and rh-activin A were examined in immature female Sprague Dawley-derived rats after iv and sc injection of the drugs. After iv administration of rh-inhibin A (120 micrograms/kg), the serum concentrations were described by a biexponential equation. The weight-normalized clearance was 21.3 ml/min.kg, and the initial (t1/2 alpha) and terminal (t1/2 beta) half-lives were 2.9 min and 37.9 min, respectively. Subcutaneous administration of 120 micrograms/kg rh-inhibin A resulted in a peak serum concentration of 10.6 ng/ml at 30.8 min after injection. Approximately 24% of the sc administered material was absorbed. Serum concentrations of rh-activin A also declined biexponentially after iv injection of the drug (120 micrograms/kg). The clearance of rh-activin A was 5.1 ml/min.kg, the t1/2 alpha was 6.1 min, and the t1/2 beta was 46.3 min. The peak serum concentration of rh-activin A (104.7 ng/ml) was achieved 24.7 min after sc delivery of the drug. The bioavailability of the sc dose was 38%. Iodinated rh-inhibin A and rh-activin A were used to examine the serum forms and metabolites of the drugs. [125I]rh-inhibin A and [125I]rh-activin A associated with two serum-binding proteins. Within 2 min of iv injection, the labeled hormones bound follistatin and alpha-2-macroglobulin. Even though rh-inhibin A and rh-activin A are structurally similar and appear to bind to the same serum proteins, their disposition in the immature rat differ.

Identification and characterization of binding proteins for inhibin and activin in human serum and follicular fluids.

Inhibins and activins are produced by a variety of tissues and may have important endocrine and paracrine roles in development, reproduction, and hematopoiesis. However, little is known regarding the physical properties or concentrations of inhibin and activin in biological fluids. Binding proteins for inhibin or activin in serum or at production or target sites may have important implications for restricting the bioactivity of these hormones and may alter the immunoreactivity of these molecules in biological fluids. The objective of this study was to identify inhibin- and activin-binding proteins in human serum (HS) and follicular fluid (hFF) and determine the ability of these proteins to alter biological or immunological activity. In HS, [125I]activin and inhibin bound to a protein identified as alpha 2-macroglobulin (alpha 2M) using three criteria: 1) [125I]inhibin and activin bind purified alpha 2M, but not several other serum proteins tested; 2) complexes formed by [125I]inhibin and activin in HS and in the presence of purified alpha 2M elute with similar retention times on HPLC; and 3) preadsorption of HS with alpha 2M antiserum inhibits inhibin and activin binding to this protein while antiserum directed against follistatin or other serum proteins had no effect. A small amount of a lower mol wt [125I]activin-follistatin complex was also found in HS. This complex eluted with a retention time similar to that of activin bound to purified porcine follistatin. Binding of inhibin to follistatin could not be detected in HS. In contrast, follistatin was the major binding protein of both activin and inhibin in hFF. Concentrations up to 100 micrograms/ml purified alpha 2M had no effect on the bioactivity or immunoreactivity of either inhibin or activin. In contrast, follistatin inhibited both activin-stimulated pituitary FSH release and K562 hemoglobin production as well as antiserum binding in a specific activin-A immunoassay. Follistatin did not interfere with inhibin immunodetection. These data indicate that two inhibin- and activin-binding proteins are present in different relative amounts in HS and hFF, alpha 2M, the primary binding protein in HS, did not alter inhibin or activin bio- or immunoactivity under the conditions of these experiments, while follistatin, the major binding protein in hFF, may mask activin's bio- and immunoactivities.

Interleukin-1 stimulates glucose transport in rat adipose cells. Evidence for receptor discrimination between IL-1 beta and IL-1 alpha.

The effect of interleukin 1 (IL-1) on glucose transport activity in isolated rat adipose cells was examined. IL-1 beta stimulated 3-O-methylglucose (3OMG) transport in a time and dose dependent manner. This effect appears to be due to increased maximal transport velocity (Vmax) of the carrier. Addition of insulin and IL-1 beta resulted in an additive stimulation of transport, suggesting different mechanisms. IL-1 alpha had no effect on glucose transport. Glu-4, a relatively inactive IL-1 beta analogue in most cells, stimulated glucose uptake in a time and dose dependent manner with kinetics indistinguishable from those of IL-1 beta.

Effect of manganese deficiency on insulin binding, glucose transport and metabolism in rat adipocytes.

The effect of manganese deficiency on insulin binding, glucose transport and metabolism in isolated adipose cells from Sprague-Dawley rats was investigated. Offspring from Mn-sufficient female rats fed 45 micrograms Mn/g diet (control) and from Mn-deficient (Mn-) female rats fed 1 microgram Mn/g diet were used in these studies. Both basal and insulin-stimulated 3-O-methylglucose transport in isolated adipose cells was significantly lower in Mn- rats, averaging 40% and 50% of control values, respectively. Kinetic analysis of glucose transport demonstrated a lower maximal transport velocity (Vmax) for glucose in adipose cells from Mn- rats compared to controls. No differences in the Km for glucose uptake were observed between the two groups. Insulin-stimulated glucose oxidation to CO2 and conversion to triglycerides was lower in isolated adipose cells from Mn- rats compared to controls. Mn- animals had fewer insulin receptors per cell compared to controls, although no differences in insulin receptor affinity were observed between the two groups. These data suggest that Mn deficiency affects glucose transport and metabolism in the adipose cell. The apparent defect lies distal to the insulin receptor and probably reflects a decreased number of glucose transporters in adipose tissue of Mn- rats.

Insulin resistant glucose transport activity in adipose cells from the SHR/N-corpulent rat.

Young male obese (cp/cp) and lean (cp/+ or +/+) littermates of the SHR/N-corpulent (cp) strain were fed purified diets containing 54% carbohydrate as either sucrose or cooked starch for 12 wk. A significant effect of phenotype (obese greater than lean) was observed on body weight, epididymal fat pad weight and fat cell size. A diet effect (sucrose greater than starch) was observed on body weight, fat pad weight, and fat cell size. No effect of phenotype or diet was observed on basal 3-O-methylglucose transport in isolated adipose cells. However, insulin-stimulated glucose uptake was decreased 70-80% in isolated adipose cells from obese SHR/N-cp rats. No effect of diet on insulin-stimulated glucose uptake was observed in obese SHR/N-cp rats. Scatchard analysis of insulin binding data demonstrated no differences in the dissociation constant (KD) for the insulin receptor:insulin complex. However, obese rats exhibited a decreased number of insulin receptors compared to lean SHR/N-cp rats. These data demonstrate that the obese SHR/N-cp rat exhibits insulin-resistant glucose transport. This altered insulin sensitivity may be one factor contributing to the development of noninsulin-dependent diabetes mellitus in these animals.

Mechanism of decreased insulinogenesis in manganese-deficient rats. Decreased insulin mRNA levels.

Manganese-deficient rats exhibited seven-fold lower preproinsulin mRNA levels compared to control, as detected by dot blot hybridization of both total and poly(A)+ RNA using a preproinsulin cDNA probe. No differences in the size of the insulin mRNA were observed. Thus, decreased mRNA levels may be a major contributing factor to the decreased insulinogenesis observed in manganese-deficient rats.

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells.

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.

Rapid and effective transfer of integral membrane proteins from isoelectric focusing gels to nitrocellulose membranes.

A method describing the rapid and effective transfer of integral membrane protein from isoelectric focusing gels to nitrocellulose is described. Initial experiments were carried out with detergent-solubilized extracts of human erythrocyte membrane proteins. The effectiveness of the transfer was demonstrated by assaying for erythrocyte glucose transporter, an integral membrane protein, using specific antibodies followed by 125I-protein A and autoradiography. Several detergents including octyl glucoside, Triton X-100 and CHAPS were used in this study but only octyl glucoside effectively solubilized the glucose transporter and did not interfere with the electrotransfer of the protein. The glucose transporter separated on isoelectric focusing gels was effectively transferred after 2 h of electroblotting and was found to have an apparent pI of 6.4-6.5. These findings were substantiated by photolabeling red cell membranes with [3H]cytochalasin B in the presence or absence of D-glucose (which inhibits [3H]cytochalasin B binding to the glucose transporter) and separating the labeled proteins by two dimensional electrophoresis. With this procedure we identified a D-glucose sensitive 50-60 kDa protein focusing with an apparent pI of around pH 6.4-6.5.

Biochemical and functional heterogeneity of rat adipocyte glucose transporters.

We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing of the eluted 40-55-kDa proteins revealed two peaks of D-glucose-inhibitable [3H]cytochalasin B radioactivity focusing at pH 6.4 and 5.6 when low-density microsomes were used as the starting material. In contrast, only one D-glucose inhibitable peak, focusing at pH 5.6, was found in plasma membranes. Pretreatment of the cells with insulin led to a marked redistribution of the pH 5.6 form of the glucose transporter from low-density microsomes to plasma membranes with no effect on the pH 6.4 form of the glucose transporter. Following isolation from the isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels, both glucose transporter isoforms were shown to cross-react with an antiserum raised against the purified human erythrocyte glucose transporter. Following incubation of [3H]cytochalasin B-labeled low-density microsomal and plasma membranes with neuraminidase, the pH 5.6 transporter isoform was shifted on isoelectric focusing to a more basic pH, while the pH 6.4 isoform was not affected. These data demonstrate that: there is a heterogeneity of glucose transporter species in the intracellular pool while the plasma membrane transporters are more uniform in structure. The pH 5.6 glucose transporter isoform is translocated by insulin from the low-density microsomes to the plasma membrane but the pH 6.4 isoform is not sensitive to insulin. Differential sensitivity of the glucose transporter isoforms to neuraminidase suggests that the heterogeneity is at least partially due to differences in glycosylation state.

Effects of manganese deficiency on pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity and carbohydrate homeostasis in adult rats.

The activities of two liver gluconeogenic enzymes, pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK), as well as liver glycogen and plasma glucose, insulin, and glucagon were measured in first- and second-generation, manganese-sufficient (control) and manganese-deficient (Mn-) adult rats. Pyruvate carboxylase activity of first generation male Mn- rats was higher than that of controls in both the fed and fasted states. In contrast, PC activity in second generation male Mn- rats was lower than control levels. In female rats, PC activity was lower than controls in both fed, first- and second-generation Mn- rats; in the fasted state, PC activity was either the same or higher than controls. Phosphoenolpyruvate carboxykinase activity was lower in male first generation Mn- rats than in controls, but there was no difference in PEPCK activity in second-generation animals. Phosphoenolpyruvate carboxylase activity was lower in both fed and fasted Mn- female rats than in controls. Plasma insulin levels were lower in the deficient rats than in controls, whereas plasma glucagon levels were similar. Manganese-deficient rats had higher concentrations of liver glycogen than their controls. These findings provide further evidence that manganese affects carbohydrate homeostasis; however, the response of the animal to manganese deficiency depends on the parameter studied and the timing of the deficiency.

Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in developing rats: effect of manganese deficiency.

The activities of two liver gluconeogenic enzymes, pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) as well as plasma glucose were measured in manganese-sufficient and manganese-deficient rats from birth to 30 d of age. Initial (d 0) PC activity was similar in the two groups. PC activity increased 1.5-fold in control pups and 2.4-fold in manganese-deficient pups from d 0 to 3 postnatally. PEPCK activity increased 1.5-fold in control pups and 2.2-fold in manganese-deficient pups from d 0 to 3. By d 8, the activity of PEPCK in manganese-deficient pups was 60% of control levels. Compared to control pups, plasma glucose concentration was lower in manganese-deficient pups on d 1 and 2, coinciding with a period of high neonatal mortality. These findings suggest that glucose homeostasis in the newborn may be compromised by manganese deficiency and may support the concept that changes in cellular manganese concentration may be important in the regulation of carbohydrate metabolism.

Dynamics of insulin and glucagon release in rats: influence of dietary manganese.

The effect of manganese on endocrine pancreatic function was examined in manganese-sufficient (control) and manganese-deficient (Mn-) Sprague-Dawley rats. Pancreatic insulin release was lower (P less than 0.05) in Mn- rats than in controls in response to both a 300 mg/dl and a 100 mg/dl glucose stimulus. The 300 mg/dl glucose stimulus induced the synthesis of 19.4 micrograms insulin/g pancreas in control rats. Additionally, no appreciable intracellular degradation of insulin occurred over an 80-min perfusion period. By contrast, in Mn- rats, there occurred an intracellular insulin degradation amounting to 7.8 micrograms/g pancreas. This enhanced degradation was partially compensated by a net insulin synthesis of only 3.4 micrograms insulin/g pancreas. Initial (min 1-3) insulin release by Mn- rats in response to 10 mM arginine was lower (P less than 0.05) than that observed in controls. Pancreatic glucagon release in response to 10 mM arginine was not affected by manganese deficiency. These findings demonstrate that manganese deficiency results in depressed pancreatic insulin synthesis and enhanced degradation. These factors may be responsible for the abnormal carbohydrate metabolism observed in Mn- animals.

Effects of high doses of manganese on carbohydrate homeostasis.

The effects of manganese (Mn) toxicity on carbohydrate homeostasis was examined in Sprague-Dawley and Osborne-Mendel rats. Mn injection was followed by increases in Mn concentration in both liver and pancreas. Concentrations of Mn in the pancreas increased more rapidly than in the liver. Plasma insulin levels decreased, plasma glucose levels increased, and a spike in glucagon concentration was observed following Mn injection. Increases in blood glucose in response to Mn injection were also observed in 24- and 48-h fasted rats, although the magnitude of the increase was less than that observed in fed rats. Both strains of rats appeared to respond similarly to Mn injection. The present studies demonstrate that acute Mn injection can affect glucose homeostasis. These effects may be mediated through altered endocrine pancreatic function.