Relationship between erythrocyte GLUT1 function and membrane glycation in type 2 diabetes.

This paper investigates the effect of glycation on glucose transport in erythrocytes. Glucose transporter function, numbers and erythrocyte phosphorylation rates are simultaneously studied using 30 Caucasian patients with diabetes and 30 Caucasian control volunteers (mean +/- SD where P < or = 0.05; age 48 +/- 8 vs. 45 +/- 8 years [ns]; body mass index [BMI] 31 +/- 7 vs. 27 +/- 5 [P=0.035]; blood glucose 12 +/- 7 vs. 5 +/- 0.6 mmol/L [P=0.001]; HbA1c 8 +/- 2 vs. 5 +/- 0.3% [P=0.0001]; fructosamine 336 +/- 64 vs. 237 +/- 16 micromol/L [P=0.0001]; disease duration 13 +/- 11 years, respectively). Significant differences were found for glucose transporter function, with 3-O-methylglucose uptake rates (108 +/- 49 vs. 146 +/- 55 micromol/L/sec/10(12) cells [P=0.010]); D-glucose influx (64 +/- 30 vs. 117 +/- 45 micromol/L/sec/10(12) cells [P=0.0001]); and D-glucose net transport (31 +/- 22 vs. 74 +/- 55 micromol/L/sec/ 10(12) cells [P = 0.0001]). No differences were found for phosphorylation rates using 2-deoxyglucose (33 +/- 17 vs. 38 +/- 12 micromol/L/sec/10(12) cells [P=0.194]). The number of functional transporters using cytochalasin B studies measured via B(max), was not found to be significantly different between the groups (195 +/- 139 vs. 264 +/- 174 [P=0.206]). However, K(d) was lower for those with diabetes, suggesting higher binding affinity (12 +/- 11 vs. 32 +/- 25 nmol/L [P=0.006]). A negative correlation between HbAlc and D-glucose influx involving both groups was found (r=-0.670, P=0.0001). Glucose transport is shown to be decreased in people who have diabetes compared to normoglycaemic volunteers, whereas the number of glucose transporters is apparently unchanged; however, affinity for binding is increased.

Relationship of glycation, antioxidant status and oxidative stress to vascular endothelial damage in diabetes.

AIMS: To examine the inter-relationships of various microvascular pathogenic mechanisms in diabetic patients. METHODS: Patients with diabetes (n = 18) and non-diabetic subjects (n = 18) were studied. RESULTS: Blood markers of glycaemic control and glycation differed between the two groups (glucose 10.9 +/- 7.6 vs. 4.7 +/- 0.63 mmol/l, p < 0.01; HbA1c 7.0 +/- 1.3 vs. 4.5 +/- 0.3%, p < 0.001; glycated LDL 8.8 +/- 2.5 vs. 6.1 +/- 1.2%, p < 0.001) but plasma antioxidant status did not. LDL oxidation resistance, measured as lag time to maximum oxidation initiated by copper ions, was decreased in diabetes (58. +/- 14.3 vs. 76.3 +/- 21.5 min, p < 0.01). Both soluble intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), markers of endothelial dysfunction, were significantly higher in diabetes (ICAM 491 +/- 128 vs. 403 +/- 131 micro g/l, p < 0.05; VCAM 546 +/- 157 vs. 393 +/- 106 micro g/l, p < 0.01). Linear correlations were significant between HbA1c and lag time of LDL oxidation (r = -0.39, p < 0.05), ICAM (r = 0.40, p < 0.05) and VCAM (r = 0.38, p < 0.05). LDL oxidizability correlated with vitamin C (r = 0.51, p < 0.01) but not any adhesion molecule. In multivariate analysis, both ICAM and VCAM correlated with HbA1c only (r(2) = 0.16, F = 6.3, p < 0.01; r(2) = 0.14, F = 5.4, p < 0.01 respectively). CONCLUSION: In diabetes, glycation, tissue oxidation and endothelial function are all abnormal and predisposing to microvascular complications but interrelationships are complex with glycation appearing most direct.

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma.

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.

Synthetic cytokines containing interleukin-3 exert potent megakaryopoietic activity.

The shared properties of haematopoietic cytokines and their receptors have enabled the genetically engineered construction of several synthetic cytokines with increased haematopoietic activity and/or more desirable pharmacological characteristics. Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic or autologous bone marrow/blood stem cell transplantation after myeloablative therapy including total body irradiation. Several in vitro, in vivo and preliminary clinical studies have demonstrated the efficacy of synthetic cytokines containing interleukin-3 in accelerating platelet recovery after radiotherapy-induced myelosuppression, enhancing G-CSF-mobilisation of CD34 positive cells for transplantation and increasing the ex-vivo expansion of myeloid and megakaryocytic progenitor cells. More randomised controlled clinical trials are needed to study the efficacy of the pre-transplant platelet mobilisation and the acceleration of the post-transplant platelet recovery. This also applies to cohort in vitro studies for expanding the production of CD41+ megakaryocytes from human bone marrow, mobilised peripheral blood and cord blood CD34 positive cells using myelopoietin as the only accepted synthetic cytokine containing interleukin-3.

Mechanisms mediating the inhibitory effect of all-trans retinoic acid on primitive hematopoietic stem cells in human long-term bone marrow culture.

All-trans retinoic acid (RA) has generally been found to stimulate late committed (colony-forming unit- granulocyte, macrophage [CFU-GM]) and inhibit early (CFU-Blast) normal human myeloid progenitor cells. The present study provides the first evidence that the pharmacological concentration of 1 microM RA, exerts an inhibitory effect on the proliferation of functional human primitive hemopoietic stem cells (cobblestone area-forming cell [CFAC]) in long-term bone marrow cultures. Treatment of four-week confluent bone marrow culture with 1 microM RA for five days significantly reduced week 4 CAFC from 88 +/- 10 in control cultures to only 52 +/- 12 per 10(5) cells, p < 0.01. Quantitative enzyme-linked immunosorbent assay measurement of interleukin 6 (IL-6) and IL-11 produced from the four-week bone marrow stroma culture revealed only a slight and moderate increase of IL-6 and IL-11 production after treatment with RA. On the other hand, treatment with RA profoundly increased the soluble receptor gp130 released from the four-week bone marrow stroma by 7.5-fold from only 145 +/- 2.1 pg per ml in control cultures to 1,069.9 +/- 3.8 pg per ml in RA-treated cultures. A similar marked increase in the soluble adhesion molecules ICAM-1, and to a lesser extent VCAM-1, released from the four-week bone marrow stroma was observed after RA treatment. IL-6 has been implicated in the inhibitory effect of RA in several human hemopoietic and nonhemopoietic cells. The common transducing signal chain gp130, for all receptors of the IL-6 cytokine family, is expressed in most primitive human hemopoietic CD34(+) cells and its signaling was shown to synergize with other hemopoietic cytokines to expand primitive human hemopoietic stem cells. Recently, soluble gp130 was shown to be a natural potent antagonist of the human IL-6 cytokine family by binding the ligand and thereby reducing its bioavailability. The profound and rapid 7.5-fold increase in the natural antagonist of human IL-6 cytokine family after RA treatment could abrogate the gp130 signaling required for proliferation and/or expansion of human primitive hemopoietic stem cells and lead to the observed inhibitory effect of RA on CAFC. Both adhesion molecules VCAM-1 and ICAM-1 mediate human hemopoietic stem cell adhesion to marrow stroma. The present significant increase in the soluble form of these adhesion molecules after RA treatment could exert a significant antagonist effect on their function and hence may impair CAFC adhesion to marrow stroma. In conclusion, the RA inhibitory effect on the proliferation of primitive human hemopoietic stem cells could be mediated through: A) an impaired hemopoietic stem cell adhesion due to the significant increase in soluble adhesion molecules released from the marrow stroma after RA treatment, and B) a significantly reduced gp130 signaling that is necessary for stem cell proliferation due to the natural antagonistic effect of the profoundly increased level of soluble gp130 released from the marrow stroma after treatment with RA.

Retinoic acid suppresses interleukin 6 production in normal human osteoblasts.

Systemic long-term retinoid therapy for chronic skin diseases significantly reduced bone turnover markers within days and led to bone abnormalities. Retinoic acid (RA) plays a key role in the regulation of mouse bone cell proliferation, differentiation and functions. Meanwhile, there is little information of RA effect on human osteoblast and osteoclast cell development and function. Interleukin 6 (IL-6) is a pleiotropic cytokine with profound effects on bone metabolism. Thus, the present study examined the RA effect on cell differentiation, alkaline phosphatase and osteocalcin production as well as IL-6 production in normal human osteoblasts. The number of large differentiated osteoblast cells decreased in RA-treated cultures P<0.05. The production of bone specific markers, alkaline phosphatase and osteocalcin, was also reduced in RA-treated cultures. Normal human osteoblasts produced 31.0+/-4.8 pg IL-6 per ml in control cultures. Within 24 h, RA at all four concentrations reduced Il-6 production from normal human osteoblasts. The pharmacological concentration of 10(-5) M RA suppressed 90% of IL-6 production. The present study shows for the first time that RA profoundly inhibits IL-6 production in normal human osteoblasts within 24 h and in a dose-dependent manner. RA was shown previously to inhibit IL-6 production in several other normal and malignant human cell types. The associated decrease in osteoblast cell differentiation, alkaline phosphatase and osteocalcin production could result from the rapid RA-inhibition of IL-6 production. Thus, RA inhibition of IL-6 production in normal human osteoblasts may contribute to the bone abnormalities seen after systemic long-term retinoid therapy in some patients.

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts.

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.

Fluoride stimulates [3H]thymidine incorporation and alkaline phosphatase production by human osteoblasts.

The effect of sodium fluoride on alkaline phosphatase (ALP) release and [3H]thymidine uptake by human osteoblasts in culture was investigated. Sodium fluoride stimulated both ALP release and [3H]thymidine uptake at concentrations of sodium fluoride greater than 250 mumol/L. This stimulation was similar in magnitude to that induced by 1,25-dihydroxycholecalciferol. The fluoride-induced increase in ALP was inhibited by verapamil, a calcium channel blocker. We conclude that sodium fluoride stimulates osteoblasts to proliferate and to release ALP. This stimulation by fluoride is dependent on calcium influx. Fluoride-induced stimulation of human osteoblasts may be relevant to its effect in enhancing bone formation in patients with osteoporosis.

86Rb(K) influx and [3H]ouabain binding by human platelets: evidence for beta-adrenergic stimulation of Na-K ATPase activity.

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), 86Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific [3H]ouabain binding by the human platelet. This 86Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active 86Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active 86Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

Diphosphonates inhibit human osteoblast secretion and proliferation.

In view of the beneficial effect of diphosphonates in Paget's disease and their suppressive effect on alkaline phosphatase, the action of three diphosphonates (aminohydroxypropylidene diphosphonate [APD], clodronate, and didronel) on cultured human osteoblasts in vitro was investigated. All three inhibited basal and 1,25(OH)2 cholecalciferol-stimulated alkaline phosphatase secretion and [3H]thymidine uptake by osteoblasts. It is concluded that diphosphonates consistently inhibit human osteoblasts through a direct action in addition to their known effect on inhibiting bone resorption and that these actions together may form the basis of their beneficial effect in vivo in patients with Paget's disease.

The effect of indomethacin and aspirin on alkaline phosphatase secretion and [3H]thymidine incorporation by human osteoblasts.

Since it has been suggested that long-term treatment with indomethacin and other non-steroidal anti-inflammatory drugs (NSAIDs) may result in destructive changes in the hip joint, we have examined the effect of indomethacin and aspirin on the secretory and mitotic activity of human osteoblasts in culture. Both indomethacin and aspirin inhibited the secretion of alkaline phosphatase and the uptake of [3H]thymidine by osteoblasts in a dose-dependent fashion at therapeutic concentrations. Both drugs also inhibited insulin- and 1,25-dihydroxycholecalciferol-stimulated alkaline phosphatase production and [3H]thymidine uptake by human osteoblasts. It is concluded that indomethacin and aspirin, and possibly other NSAIDs, may have an inhibitory effect on osteoblast function.

The effect of polyunsaturated fatty acids on rat adipocyte lipogenesis: the role of protein kinase C, calcium and calmodulin.

Linolenic acid, a polyunsaturated fatty acid, has an insulin synergizing stimulatory effect on adipocyte lipogenesis. Since phorbol esters are also known to exert a similar effect through the activation of protein kinase C, and since they have fatty acid moieties, we investigated whether linolenic acid exerted its stimulatory effect through protein kinase C activation and whether calcium was involved in this mechanism. Our experiments show that H7, an inhibitor of protein kinase C; verapamil, a calcium blocker; and calmodulin inhibitors inhibited basal, insulin- and linolenic acid-stimulated lipogenesis. They also negated the insulin synergizing effect of linolenic acid. We conclude that linolenic acid, and possibly other unsaturated fatty acids, exert their stimulatory effect through stimulation of protein kinase C, calcium entry and calmodulin activation. These three processes are also important in maintaining basal lipogenesis.

Ethanol and its novel metabolites inhibit insulin action on adipocytes.

The effects of ethanol, acetaldehyde, and the two novel metabolites of ethanol 2,3-butanediol and 1,2-propanediol on basal and insulin-stimulated adipocyte lipogenesis and glucose oxidation in vitro were investigated. Whereas ethanol and acetaldehyde inhibited both processes at concentrations far greater than those found in alcoholic subjects, the two diols were extremely potent inhibitors of basal and insulin-stimulated adipocyte metabolism at concentrations far below those observed in alcoholic subjects. The incorporation of labeled glucose into the fatty acid moiety and glucose oxidation were inhibited at lower concentrations (0.25 microM) than those required to inhibit the incorporation of the labeled glucose into glycerol. The diols are therefore potent inhibitors of basal and insulin-stimulated adipocyte metabolism. This effect may be relevant to the pathogenesis of insulin resistance in alcoholic subjects.

Similarities between the insulin-like stimulatory effect of human IgG and NSILP.

Since the insulin-like stimulatory effect of human IgG on adipocyte lipogenesis, exerted through its Fc moiety, is not neutralized by anti-insulin antisera, IgG may contribute significantly to the non-suppressible insulin-like activity (NSILA) of plasma. 91% of NSILA has been shown previously to be associated with a high mol. wt. protein (NSILP). The purpose of this investigation was to assess whether IgG and NSILP have similar stimulatory effects on adipocyte lipogenesis and whether this effect can be neutralized by preincubation with gamma-chain specific anti-IgG antiserum; whether IgG stimulates 35S-sulphate uptake by porcine cartilage, known to be stimulated by insulin-like growth factors but not NSILP; and whether gamma-chain specific anti-IgG antisera precipitate IgG in a fashion similar to that with IgG preparations. Our investigations show that both IgG and NSILP have similar dose response relationships with respect to the stimulation of adipocyte lipogenesis and that both lose their adipocyte stimulating effect following preincubation with anti-IgG antiserum; neither IgG nor NSILP stimulate 35S-sulphate uptake by porcine cartilage, unlike serum somatomedin and crude NSILA-s preparations; and that gamma-chain specific anti-IgG antisera form precipitin lines with NSILP. Therefore, NSILP and IgG molecules have immunological and biological similarities; there may occur a homology between the Fc fragment of IgG and the NSILP molecule.

Human plasma fractions inhibit the action of insulin on adipocytes and somatomedin on cartilage.

Since human immunoglobulins exert an insulin-like stimulatory effect on adipocyte lipogenesis at concentrations markedly lower than those found in vivo, and since human serum or plasma are only midly stimulatory, we predicted that human serum probably contains an inhibitor of adipocyte lipogenesis. Supernatant preparations, obtained from the precipitation of immunoglobulins from plasma in 2.5 mol/l ammonium sulphate, were extensively dialysed and tested for their activity on bioassay systems commonly used for measuring insulin. The supernatants produced a marked inhibition of basal and insulin- or IgG-stimulated lipogenesis and glucose oxidation by adipocytes at protein concentrations of 10 mg/l. The supernatants were further purified through ultrafiltration to demonstrate two main inhibitory fractions, 10 to 30 K and 30 to 50 K, which again produced marked inhibition of basal and insulin- or IgG-stimulated adipocyte lipogenesis and glucose oxidation. These fractions were then tested for basal and serum somatomedin-stimulated 35S sulphate uptake by porcine cartilage: both basal and serum somatomedin-stimulated 35S uptake were significantly inhibited (p less than 0.01). Therefore, normal human serum contains at least two peptides which are markedly inhibitory to glucose metabolism and insulin action on adipocytes and 35S transport and somatomedin action on cartilage.

Stimulatory effect of immunoglobulins and Fc fragments on glucose oxidation and glucose transport by adipocytes.

Following our demonstration that human IgG, its Fc fragments and IgM stimulate lipogenesis in adipocytes, we embarked on a study to investigate whether these proteins stimulate the oxidation and transport of glucose by adipocytes, and whether such a stimulation is mediated through the insulin receptor. Using a simplified method for the measurement of [14C]-carbon dioxide produced from [U-14C]-glucose by rat adipocytes, we demonstrated that both IgG, Fc fragments and IgM produced a dose-dependent stimulation of oxidation of glucose by adipocytes. These proteins also produced a stimulation of 3-o-methylglucose uptake by adipocytes. IgG, Fc Fragments and IgM did not, however, alter specific binding of insulin to adipocytes. These data provide further evidence that human IgG and IgM may have a role in regulating metabolic activity of adipose tissue. These effects are exerted independently of the insulin receptor and are probably mediated through the interaction of its Fc fraction with putative Fc receptors on the adipocyte membrane. A similar interaction may be important in the regulation of metabolic activity of other cells with Fc receptors.