Immunological detection of glycated proteins in normal and streptozotocin-induced diabetic rats using anti hexitol-lysine IgG.

A polyclonal antibody specific for the Amadori compound, a product of an early stage of the Maillard reaction, was raised in rabbits by immunization with hexitol-lysine (1-glucitol-lysine or 1-mannitol-lysine) coupled with various carrier proteins. The affinity purified antibody has a high titre and preferentially recognizes the glucose adduct, in the presence of sodium borohydride, as judged on enzyme-linked immunosorbent assay as well as immunoblot analysis. The glycated proteins (Amadori products) in various tissues of normal and streptozotocin-induced diabetic rats were examined by immunoblot analysis. In diabetic conditions, kidney, liver, lens, brain and lung proteins are more susceptible to glycation than other tissue proteins. Heart, spleen, adrenal gland and muscle proteins exhibit similar extents of glycation in both normal and diabetic conditions. This is the first demonstration of a specific antibody against the Amadori compound being raised with a synthetic compound, and of the tissue distribution of glycated proteins in normal and diabetic conditions. The antibody was very useful for in vitro and in vivo experiments on the Maillard reaction.

Age-associated increase of basal corticosterone levels decreases ED2high, NF-kappaBhigh activated macrophages.

The proportion of cells with a high density of ED2 (ED2high cells) in peritoneal cells from old rats was significantly lower than that from young rats. The expression of major histocompatibility complex class II (MHC class II) molecules, the antigen presentation, production of interleukin (IL)-1beta and IL-6, and nuclear factor-kappaB activity in ED2high cells were markedly higher than those in cells with a low density of ED2 (ED2low cells), although no significant difference was observed in the expression of MHC class II molecules and the antigen presentation between ED2high cells from young and old rats. Meanwhile, basal corticosterone concentration in serum and glucocorticoid (GC) receptor mRNA expression in peritoneal cells increased significantly in old rats. The proportion of ED2high cells was increased by adrenalectomy in young rats. Furthermore, nuclear translocation of GC receptor was observed in ED2low cells, whereas GC receptor was detected in cytoplasmic extracts from ED2high cells. These results suggest that the decrease in functional ED2high macrophages with age results in the age-associated decline of immune responses, which is regulated, in part, by the basal GC concentration.

Glucocorticoid-mediated generation of suppressor macrophages with high density Fc gamma RII during acute cold stress.

Acute cold stress induces adherent cells with suppressor function, resulting in immunosuppression. Glucocorticoids (GC) are known as an inhibitor of the immune system and are increased by stress through stimulation of the hypothalamic-pituitary-adrenal axis. To elucidate the mechanisms underlying acute cold stress-induced immunosuppression, functions and surface phenotypes of murine peritoneal cells of the monocyte/macrophage lineage from acute cold-stressed mice (5 C for 3 or 24 h) in addition to the role of GC in the immunomodulation were investigated. Flow cytometric analysis revealed that the proportion of MAC-1+ cells with a high density of Fc gamma RII (Fc gamma RIIbright cells) was markedly increased in the peritoneal exudate cells from acute cold-stressed mice. These Fc gamma RIIbright cells were also stained with F4/80, a monoclonal antibody directed specifically against the mouse mature macrophages. The prominent suppressor activity for Concanavalin A (Con A) responses of control spleen cells was found in Fc gamma RIIbright cells, whereas MAC-1+ cells, with a low density of Fc gamma RII (Fc gamma RIIdull cells), from the stressed mice did not suppress the Con A responses. Fc gamma RIIbright cells from control mice also suppressed the Con A responses; the inhibitory effect was considerably less than that of cells from acute cold-stressed mice. As was anticipated, serum corticosterone levels were markedly increased in acute cold-stressed mice. In addition, expression of GC receptor messenger RNA was observed in Fc gamma RIIbright cells from these mice. The increase in Fc gamma RIIbright cells in peritoneal exudate cells caused by acute cold stress was inhibited by adrenalectomy or administration of a saturating amount of the GC antagonist RU 38486 (mifepristone). On the contrary, administration of the GC agonist, dexamethasone, markedly increased the proportion of Fc gamma RIIbright cells in peritoneal exudate cells of control mice. These results suggest that the generation of Fc gamma RIIbright suppressor cells of monocyte/macrophage lineage by acute cold stress was mediated to a greater or lesser degree by the action of GC through the GC receptor.

Increased glycated Cu,Zn-superoxide dismutase levels in erythrocytes of patients with insulin-dependent diabetis mellitus.

Our previous study indicated that erythrocyte Cu,Zn-superoxide dismutase (Cu,Zn-SOD) undergoes glycation and inactivation in vivo (1) and in vitro (2). The aim of the present study was to assess glycated Cu,Zn-SOD in patients with insulin-dependent diabetes mellitus. Glycated Cu,Zn-SOD, which binds to a boronic acid affinity column, was measured by the enzyme-linked immunosorbent assay. The percentage of the glycated form in 25 insulin-dependent diabetic children was 40.2 +/- 8.2%; this was significantly higher than that in the normal controls (P less than 0.01). The specific activity of the glycated form in the diabetic children was 163,000 +/- 33,000 IU/mg Cu,Zn-SOD protein, significantly lower than that in controls (P less than 0.01). These data indicate that glycated and less active Cu,Zn-SOD is increased in erythrocytes of patients with insulin-dependent diabetes mellitus.

Superoxide dismutase gene expression in skeletal muscle: fiber-specific effect of age.

The influence of ageing on the expression of two superoxide dismutase (SOD) isozymes was examined in three different skeletal muscle fiber types of young (Y, 8 mo) and old (O, 25 mo) rats. Total SOD activity was increased with age in the gastrocnemius (Gas, type II(mix)) and superficial vastus lateralis (SVL, type IIb) but unchanged in the soleus (Sol, type I). The increased SOD activity in SVL was due to increased cytosolic SOD (CuZn SOD), whereas both mitochondrial (Mn SOD) and CuZn SOD activities were increased in Gas. In Sol, Mn SOD activity was significantly increased in aged rats. Mn SOD mRNA level was significantly decreased with age in all three muscles examined, while Mn SOD protein content was not altered. Ageing did not affect CuZn SOD mRNA abundance in any of the muscles, but significantly increased CuZn SOD protein content in aged Gas and Sol. Binding of two redox-sensitive transcription factors, nuclear factor-kappaB (NFkappaB) and activator protein-1 (AP-1) was significantly decreased with age in all three muscle types. These results indicate that increased SOD activity in aged skeletal muscle is not associated with higher levels of gene transcription. Increases in Mn SOD activity seen in aged Gas and Sol are the result of post-translational modification of the enzyme, whereas increases in CuZn SOD activity during ageing may be due to both translational and post-translational control.

Effect of age on brown adipose tissue activity in the obese (ob/ob) mouse.

Brown adipose tissue (BAT), a highly thermogenic tissue in young animals, is relatively atrophied and thermogenetically quiescent (e.g. as measured by colonic temperature) in mice that are obese or old. The aim of the current study was to investigate the effect of aging (3.1 (young) versus 14.6 (old) months old) on BAT activity in lean and obese (ob/ob) mice. In young but not in old mice, BAT mass in terms of weight per unit body weight was significantly lower in obese mice than in lean mice. A significant increase in BAT mass of obese mice with age was noted in terms of weight or weight per unit body weight, probably because of a tendency to become white adipose tissue and the deposit of fat, accompanied by the lowest levels of total protein, guanosine 5'-diphosphate binding, and uncoupling protein (UCP) antigen in the mitochondria of BAT, as well as the lowest colonic temperature among the groups examined. Unlike old lean animals, the old obese (ob/ob) animals did not increase but rather decreased the expression of mRNA for UCP in the mitochondria of BAT. These findings suggest that a marked decrease in BAT thermogenic capacity and activity is noted in old obese mice, probably due to synergism of aging and obesity.

Relationship between cold tolerance and generation of suppressor macrophages during acute cold stress.

Acute cold stress induces suppressor macrophages expressing large numbers of receptors to the crystallizable fragment (Fc) portion of immunoglobulin G (MAC-1+ FcgammaRII/IIIbright cells), resulting in the immunosuppression of splenocyte mitogenesis. The generation of MAC-1+ FcgammaRII/IIIbright cells is mediated by the action of glucocorticoids (GCs) through the GC-receptor. In the present study, the generation of MAC-1+ FcgammaRII/IIIbright cells in peritoneal exudate cells was closely related to the decrease of rectal temperature during 3-day exposure to 5 degrees C. We next investigated the effects of improved cold tolerance on the generation of MAC-1+ FcgammaRII/IIIbright cells during acute cold stress. Mice were adapted to cold by exposure to 5 degrees C for 3 wk (cold-acclimated mice) and then reexposed to 5 degrees C for 3 h (acute cold stress) after living at 25 degrees C for 24 h. The rectal temperature of cold-acclimated mice was not decreased by the acute cold stress. In addition, the proportion of MAC-1+ FcgammaRII/IIIbright cells in peritoneal exudate cell population from cold-acclimated mice was unaffected by the acute cold stress. The cold acclimation significantly attenuated the increases in serum corticosterone levels and the expression of the GC-receptor mRNA on peritoneal exudate cells in response to acute cold stress. These results suggest that the altered GC response to acute cold stress by the improvement of cold tolerance inhibits the generation of suppressor macrophages during acute cold stress.

Effects of swimming training on three superoxide dismutase isoenzymes in mouse tissues.

The purpose of the present study was to investigate the effects of swimming training on the changes in three superoxide dismutase (SOD) isoenzymes in mice. The trained mice underwent a 6-wk swimming program (1 h/day, 5 days/wk) in water at 35-36 degrees C. Immunoreactive extracellular SOD (EC-SOD), copper- and zinc-containing SOD (CuZn-SOD), and manganese-containing SOD (Mn-SOD) contents and their mRNA abundance were determined in serum, heart, lung, liver, kidney, and gastrocnemius muscle. EC-SOD content in liver and kidney was significantly increased with training. After training, CuZn-SOD content rose significantly only in kidney but decreased significantly in heart, lung, and liver. Mn-SOD content showed a significant increase in lung, kidney, and skeletal muscle but a significant decrease in liver. In most tissues, however, the changes in SOD isoenzyme contents were not concomitant with those in their mRNA levels. The results obtained thus suggest that, except for kidney, the responses in mouse tissues of three SOD isoenzymes (protein levels and mRNA abundance) to swimming training are different and that kidney may be one of the most sensitive organs to adapt to oxidative stress during physical training, although the mechanism remains vague.

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice.

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-alpha and interleukin-1beta contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.

Is insulin-like growth factor I involved in brown adipose tissue enlargement?

During cold exposure, the expression of insulin-like growth factor I (IGF-I) mRNA in rat brown adipose tissue (BAT) increased significantly with the increase in the BAT weight. In addition, the blood plasma from cold-acclimated (CA) rats enhanced the proliferation of brown adipocyte precursor cells in primary culture and the expression of IGF-I mRNA in them compared to those from warm-acclimated rats. The cell proliferation was considerably inhibited by anti-growth hormone (GH) antibody. These results suggest that IGF-I produced by brown adipocytes may play a role in BAT enlargement during cold acclimation. It is probable that some factors (including GH) concerned with the proliferation of brown adipocyte precursor cells are involved in the blood plasma from CA rats.

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase.

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.

Swimming training prevents generation of suppressor macrophages during acute cold stress.

PURPOSE: Acute cold stress induces suppressor macrophages expressing large numbers of receptors to Fc portion of immunoglobulin G (MAC-1+ Fc gammaRII/IIIbright cells), resulting in suppression of splenocyte mitogenesis. The generation of MAC-1+ Fc gammaRII/IIIbright cells is partly mediated by increased glucocorticoid levels during acute cold stress. The aim of the current study was to investigate the effect of swimming training on the generation of the MAC-1+ Fc gammaRII/IIIbright suppressor macrophages by acute cold stress. METHODS: The trained mice underwent a 6-wk endurance swimming training (5 times/wk) in water at 35-36 degrees C for 90 min. The swimming training significantly increased brown adipose tissue mass, suggesting improved cold tolerance. Actually, when the swimming-trained mice were exposed to 5 degrees C for 3 h (acute cold stress), the rectal temperature was not decreased. The proportion of MAC-1+ Fc gammaRII/IIIbright cells in peritoneal exudate cells from swimming-trained mice was significantly lower than that from control mice. In addition, the proportion of MAC-1+ Fc gammaRII/IIIbright cells in peritoneal exudate cell population from swimming-trained mice was unaffected by the acute cold stress. The swimming training significantly attenuated the increases in serum corticosterone levels in response to acute cold stress. These results suggested that swimming training not only improves cold tolerance but also inhibits the generation of suppressor macrophages under acute cold stress as well as under normal conditions.

Characteristics of plasma extracellular SOD in burned patients.

The aim of this study was to examine three types of superoxide dismutase (SOD) in plasma after burns, and especially to clarify the characteristics of plasma extracellular SOD (EC-SOD) in burned patients. A total of 71 blood samples were collected from 18 patients on arrival, day 1, day 3 and day 5 after burns. We measured three types of SOD (Mn, Cu/Zn, EC) in plasma using ELISA, and the relationships among the three types of SOD concentrations were examined. We next analyzed the characteristics of plasma EC-SOD using stepwise multivariate regression analysis. Any plasma SOD isoenzyme concentration measured after burns was beyond the normal range and EC-SOD accounted for the major part of plasma SODs. EC-SOD and Cu/Zn-SOD were positively correlated, whereas Mn-SOD was not related to the other SODs. Also, plasma EC-SOD was significantly related to existence of inhalation injury, %TBSA and age, respectively. The plasma EC-SOD might therefore play some roles in the pathophysiology of burned patients.

Extracellular superoxide dismutase following cerebral ischemia in mice.

We describe the changes in extracellular superoxide dismutase (EC-SOD) following cerebral ischemia in mice. Mice were subjected to transient forebrain ischemia and reperfusion. The measurements of EC-SOD using ELISA showed increased brain EC-SOD after 24 h of reperfusion. The immunohistochemical examination showed that EC-SOD immunoreactivity in cortical and striatal capillary wall was conspicuous after 3 h. EC-SOD immunoreactivity was also noted in cortical neurons after 24 h. Northern blot analysis showed an increased EC-SOD mRNA expression in the brain after 24 h. In situ hybridization study demonstrated no mRNA expression of EC-SOD following ischemia and reperfusion in the capillary wall. These findings suggest that serum EC-SOD might accumulate on brain endothelial cells, while cortical neurons produce EC-SOD themselves after cerebral ischemia with reperfusion.

[Studies on growth of brown adipose tissue by means of cell culture and angiogenesis in vitro techniques].

To elucidate the mechanism of brown adipose tissue (BAT) enlargement, we investigated the effects of norepinephrine (NE) and insulin on the in vitro growth of rat brown adipose cells (RBAC) and the capillary growth in angiogenesis in vitro using co-culture of bovine capillary endothelial cells (BCEC) and rat smooth muscle cells. In the primary cell culture, NE significantly enhanced the growth of RBAC in the range of 10(-9)-10(-5)M, whereas it did not stimulate the growth of BCEC. Insulin showed the same trend. Moreover, both NE and insulin appeared to increase the expression of mRNA for basic fibroblast growth factor (bFGF), which is a potent angiogenic factor, in RBAC. At 4 h after NE stimulation, the bFGF mRNA expression was considerably increased but it decreased markedly after 16 h. These results suggest that the bFGF mRNA expression in RBAC is quickly simulated by NE, wih resulting bFGF production. Actually, bFGF stimulated the RBAC growth up to about 170% of the control level. However, neither NE nor insulin stimulated the expression of the bFGF gene in BCEC. On the other hand, NE notably increased the capillary growth in vitro compared with insulin. It is thus possible that NE and insulin contribute to the growth of RBAC and endothelial cells partly through bFGF production by an autocrine mechanism, suggesting that both agonists play an important role not only in the thermogenic function of BAT but also in BAT enlargement.

Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species.

Site-specific and random fragmentation of human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was observed following the glycation reaction (the early stage of the Maillard reaction). The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro62 and His63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5 kDa) fragment. In the second step, random fragmentation occurred. The ESR spectrum of the glycated Cu,Zn-SOD suggested that reactive oxygen species was implicated in the both steps of fragmentation. The same fragmentations were seen upon exposure of the enzyme to an H2O2 bolus. Catalase completely blocked both steps of the fragmentation process, whereas EDTA blocked only the second step. Incubation with glucose resulted in a time-dependent release of Cu2+ from the Cu,Zn-SOD molecule. The released Cu2+ then likely participated in a Fenton's type of reaction to produce hydroxyl radical, which may cause the nonspecific fragmentation. Evidence that EDTA abolished only the second step of fragmentation induced by an H2O2 bolus supports this mechanism. This is the first report that a site-specific fragmentation of a protein is caused by reactive oxygen species formed by the Maillard reaction.

Effects of ageing on generation of ED2high major histocompatibility complex class II+ macrophages during cold stress.

The effects of cold stress (5 degrees C, 24 h) were investigated on the function and surface phenotype of peritoneal cells in the monocyte/macrophage lineage from young (8-10-week-old) and old (22-24-month-old) rats. The role of glucocorticoid (GC) in the immunomodulation by cold stress was also examined. The proportion of cells with a high density of ED2 (ED2high cells), expressing major histocompatibility complex (MHC) class II molecules, was significantly increased in peritoneal cells during cold stress in young, but not in old, rats. Antigen-presenting function was significantly higher in ED2high cells than in cells with a low density of ED2 (ED2low cells), thereby indicating that ED2high cells are at a functionally high level. While serum corticosterone concentration in old rats increased markedly after 3 h of cold stress, that in young rats did not vary substantially, and was followed by a significant decrease in both groups of rats after 24 h of cold stress. Administration of dexamethasone to young rats completely inhibited the increase of ED2high cells caused by cold stress. Meanwhile, the proportion of ED2high cells in young rats was significantly increased by adrenalectomy. Furthermore, nuclear translocation of a large amount of the GC receptor was observed in ED2low cells. These results suggest that cold stress enhances immune function in young rats, but not in old rats, and that the generation of ED2high cells are partly regulated by circulating GC level.