Efectos "in vivo" de Metformina sobre las alteraciones de la microarquitectura sea asociadas al Sndrome Metablico inducido por fructosa en ratas / In vivo effects of Metformin on the alterations of bone micro-architecture associated with fructose-induced Metabolic Syndrome in rats

El Sndrome Metablico (SM) se ha asociado recientemente con una disminucin en la densidad mineral sea, y con un aumento en la incidencia de fracturas osteoporticas. Recientemente encontramos que la Metformina por va oral en ratas, promueve la diferenciacin osteognica de clulas progenitoras de mdula sea e incrementa la reparacin de lesiones seas. En este trabajo evaluamos los efectos del SM inducido por Fructosa sobre la microarquitectura sea en ratas, y la modulacin de estos efectos por Metformina administrada en forma oral. Utilizamos ratas Sprague Dawley macho jvenes: C (control sin tratamiento), C+M (100mg/kg/da Metformina en el agua de bebida), F (10 % Fructosa en el agua de bebida) y F+M (Fructosa+Metformina en el agua de bebida). Los tratamientos se continuaron por 3 semanas luego de lo cual se tomaron muestras de sangre, previas al sacrificio de los animales. Se disecaron los fmures para evaluacin histomorfomtrica de la microarquitectura metafisaria por tincin con Hematoxilina-Eosina (H-E). Se observ un incremento en la glucemia y trigliceridemia en el grupo F versus el C, compatible con el desarrollo de SM. El anlisis de las metfisis femorales mostr un aumento en la densidad osteoctica trabecular para el grupo C+M (118 % del control, p<0,05). El tratamiento con Fructosa sola disminuy la densidad osteoctica (79 % del control, p<0,05), mientras que el co-tratamiento Fructosa+Metformina (grupo F+M) revirti parcialmente este descenso (88 % del control). Similarmente, el porcentaje de hueso trabecular en la metfisis femoral aument luego del tratamiento slo con Metformina (129 % respecto del control), se redujo en las ratas tratadas con Fructosa (89 % respecto del control), y fue intermedia en el grupo F+M (94 % respecto del control). Estos resultados muestran que el SM inducido por Fructosa en ratas altera la microarquitectura metafisaria femoral; y que estos efectos deletreos pueden ser parcialmente prevenidos por un tratamiento oral con Metformina.

Several clinical studies have demonstrated that the Metabolic Syndrome (MS) is associated with a decrease in bone mineral density, and with an increased risk for non-vertebral osteoporotic fractures. We have recently found that orally administered Metformin induces osteogenic effects in rats, promoting osteoblastic differentiation of bone marrow progenitor cells and increasing the repair of bone lesions. In the present work we have evaluated the effects of Fructose-induced MS on bone micro-architecture in rats, and the possible modulation of these effects by orally administered Metformin. We utilized young male Sprague-Dawley rats, divided into four groups: C (non-treated controls); C+M (100 mg/kg/day of Metformin in drinking water); F (10 % of Fructose in drinking water); and F+M (Fructose+Metformin in drinking water). After three weeks of all treatments blood samples were taken, after which animals were sacrificed by cervical dislocation under anaesthesia. Femurs were then dissected for evaluation of metaphyseal micro-architecture after Haematoxilin-Eosin staining of 5 μm histological slices of decalcified bone. In particular, osteocytic density and relative trabecular volume were determined. An increase in serum glucose and triglycerides was observed in Fructose-treated rats, in accordance with the development of MS. In rats treated with Metformin alone (group C+M), the analysis of femoral metaphyses showed an increase in trabecular osteocytic density (118 % of control [group C], p<0.05). Treatment with Fructose alone (group F) significantly decreased ostecytic density (79 % of control, p<0.05), while co-treatment with Fructose and Metformin partially reverted this decrease (group F+M, 88 % of control). Similarly, the relative trabecular volume of femoral metaphysic was increased by treatment with Metformin alone (129% of control), was reduced in Fructose-treated rats (89 % of control), and tended to revert back to control values after Fructose-Metformin co-treatment (94 % of control). These results show for the first time that (a) Fructose-induced MS in rats alters their femoral metaphysis micro-architecture; and that (b) these deleterious effects can be partially prevented by orally administered Metformin.

Metformin reverts deleterious effects of advanced glycation end-products (AGEs) on osteoblastic cells.

Advanced glycation endproducts (AGEs) are implicated in the complications of diabetes and ageing, affecting several tissues, including bone. Metformin, an insulin-sensitizer drug, reduces the risk of life-threatening macrovascular complications. We have evaluated the hypothesis that metformin can abrogate AGE-induced deleterious effects in osteoblastic cells in culture. In two osteoblast-like cell lines (UMR106 and MC3T3E1), AGE-modified albumin induced cell death, caspase-3 activity, altered intracellular oxidative stress and inhibited alkaline phosphatase activity. Metformin-treatment of osteoblastic cells prevented these AGE-induced alterations. We also assessed the expression of AGE receptors as a possible mechanism by which metformin could modulate the action of AGEs. AGEs-treatment of osteoblast-like cells enhanced RAGE protein expression, and this up-regulation was prevented in the presence of metformin. Although the precise mechanisms involved in metformin signaling are still elusive, our data implicate the AGE-RAGE interaction in the modulation of growth and differentiation of osteoblastic cells.

Family history of depression is associated with younger age of onset in patients with recurrent depression.

BACKGROUND: Genetic epidemiology data suggest that younger age of onset is associated with family history (FH) of depression. The present study tested whether the presence of FH for depression or anxiety in first-degree relatives determines younger age of onset for depression. METHOD: A sample of 1022 cases with recurrent major depressive disorder (MDD) was recruited at the Max Planck Institute and at two affiliated hospitals. Patients were assessed using the Schedules for Clinical Assessment in Neuropsychiatry and questionnaires including demographics, medical history, questions on the use of alcohol and tobacco, personality traits and life events. Survival analysis and the Cox proportional hazard model were used to determine whether FH of depression signals earlier age of onset of depression. RESULTS: Patients who reported positive FH had a significantly earlier age of onset than patients who did not report FH of depression (log-rank=48, df=1, p<0.0001). The magnitude of association of FH varies by age of onset, with the largest estimate for MDD onset before age 20 years (hazard ratio=2.2, p=0.0009), whereas FH is not associated with MDD for onset after age 50 years (hazard ratio=0.89, p=0.5). The presence of feelings of guilt, anxiety symptoms and functional impairment due to depressive symptoms appear to characterize individuals with positive FH of depression. CONCLUSIONS: FH of depression contributes to the onset of depression at a younger age and may affect the clinical features of the illness.

Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress.

BACKGROUND: The tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects. RESULTS: AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. CONCLUSIONS: These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

Effect of advanced glycation endproducts on the secretion of insulin-like growth factor-I and its binding proteins: role in osteoblast development.

In chronically uncompensated diabetes mellitus, an increase has been observed in the content of advanced glycation endproduct (AGE)-modified proteins in various tissues, including bone. This increase can lead to a local imbalance in the secretion of cytokines and growth factors, and has been implicated in the pathophysiology of the longterm complications of diabetes. We have previously shown that the proliferation and differentiation of UMR106 rat osteosarcoma and MC3T3E1 mouse calvaria-derived cell lines are regulated by AGE-modified proteins, possibly through the recognition of these AGEs by specific membrane-associated receptors. In the present study, we investigated the effects of AGE-proteins on the secretion of insulin-like growth factor-I (IGF-I) and its binding proteins (IGFBPs) by both osteoblast-like cell lines. In the case of MC3T3E1 cells, this was studied throughout their successive stages of development: proliferation, differentiation and mineralisation. For every condition, cells were incubated 24 hours with increasing concentrations of either bovine serum albumin (BSA) or AGE-BSA. IGF-I in conditioned media was separated from IGFBPs by acid gel filtration-centrifugation, and measured by radioimmunoassay. IGFBPs in conditioned media were analysed by a semi-quantitative western ligand blot. In UMR106 cells, low doses of AGE-BSA significantly decreased the secretion of both IGF-I (56% of control) and a 24 kDa IGFBP (80% of control). Results for MC3T3E1 cells, which predominantly secrete 29 kDa IGFBPs, were dependent on the stage of development. In proliferating preosteoblastic cells, AGE-BSA decreased the secretion of IGF-I (34%-37% of control) while increasing the secretion of IGFBP (124%-127% of control). On the other hand, secretion of these components of the IGF system by mature (differentiated) cells was unaffected by the presence of AGE-BSA. When these cells finally attained mineralisation, incubation with AGE-modified BSA provoked an increase both in IGFBP (131%-169% of control) and in IGF-I secretion (119%-123% of control). The presented evidence suggests that the modulation of growth and development by AGE-modified proteins, previously described for both cell lines, could be the result of an autocrine-paracrine mechanism involving the IGF-IGFBP system.

Advanced glycation endproduct-specific receptors in rat and mouse osteoblast-like cells: regulation with stages of differentiation.

Advanced glycation endproducts have been implicated in the development of diabetic complications. In addition, these products could also mediate certain bone alterations such as diabetic osteopenia. Several receptors specific for advanced glycation endproduct-modified proteins have been characterized in different cell types, contributing to the recognition and degradation of senescent proteins. In the present report, we investigated the possible presence of advanced glycation endproduct-binding proteins on osteoblast-like cells. Both UMR106 and MC3T3E1 cell lines express specific advanced glycation endproduct-binding sites, with an affinity constant between 0.4 and 1.7. 10(6) M(-1), depending on the stage of osteoblastic differentiation; and with a receptor capacity of 1.5-2.0. 10(7) sites/cell. Osteoblast-like cells were also found to participate both in the uptake and degradation of advanced glycation endproduct-modified bovine serum albumin at 37 degrees C. Radiolabelled ligand blotting studies confirmed the presence of several membrane binding proteins, with apparent molecular masses of 50, 45-40, 30, 25 and 18 kDa; the major bands corresponded to 30 and 25 kDa proteins. This study provides evidence of the presence of advanced glycation endproduct-specific binding sites, and for their regulation with the stage of differentiation, in two osteoblast-like cells in culture.

Non-enzymatic glycosylation of alkaline phosphatase alters its biological properties.

Hyperglycaemia in poorly controlled diabetic patients induces non-enzymatic glycosylation (glycation) of proteins, altering their structure and physiological bioactivity. Alkaline phosphatase (ALP) is a membrane-bound exoenzyme which faces the extracellular compartment. We have investigated the glycation of intestinal alkaline phosphatase in vitro and the consequences of such molecular modifications on certain structural and functional characteristics. The effect of glycation on alkaline phosphatase specific activity was determined after incubation of the enzyme with different sugars for various periods of time. The formation of early reversible glycation products was determined by the measurement of fructosamine levels, while the appearance of advanced glycation end products was estimated by spectrofluorometric analysis. A decrease in the specific activity of ALP was associated both with an increase in fructosamine levels and with the appearance of AGE-characteristic fluorescence. Changes in these parameters were found to depend on the incubation time, and on the concentration and glycating capability of the sugar employed. Co-incubation with aminoguanidine slowed down the appearance of protein-linked fluorescence, and additionally curbed the decrease in enzymatic specific activity. A significant correlation between the levels of ALP-fructosamine and ALP-advanced glycation end product was observed. Patterns of protein bands fractionated by SDS-PAGE were essentially identical for the nonglycated controls and the glycated samples. The electrophoretic mobility of the band of alkaline phosphatase on cellulose acetate gels increased as a function of the incubation time and the glycosylating power of the carbohydrate used. The present study provides evidence for the in vitro glycation of alkaline phosphatase, and for the consecutive alteration of its activity and structure.

A commercially viable virtual reality knee arthroscopy training system.

Arthroscopy is a minimally invasive form of surgery used to inspect joints. It is complex to learn yet current training methods appear inadequate, thus negating the potential benefits to the patient. This paper describes the development and initial assessment of a cost-effective virtual reality based system for training surgeons in arthroscopy of the knee. The system runs on a P.C. Initial assessments by surgeons have been positive and current developments in deformable models are described.

Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells.

Two different lines of osteoblast-like cells were used to investigate the effect of advanced glycation end-products of bovine serum albumin on cell proliferation and differentiation. These parameters were found to be both dose- and time-dependent. Cell proliferation remained unchanged after a 24 h incubation period, it increased after intermediate periods of incubation with advanced glycation end-products, but was found to be depressed after several days incubation. Cellular alkaline phosphatase activity followed a similar pattern: an initial increase induced by advanced glycation end-products was generally followed, after relatively long incubation periods, by a slight but significant decrease in this parameter. 45Ca2+ uptake was only significantly inhibited by advanced glycation end-products after 24 h incubation. These results suggest that advanced glycation end-products directly regulate osteoblast proliferation and differentiation in a dose and time dependent manner.

The squalestatins: inhibitors of squalene synthase. Enzyme inhibitory activities and in vivo evaluation of C3-modified analogues.

Squalestatin analogues modified at C3 were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase in vitro. While the 4,6-dimethyloctenoate ester group at C6 was maintained, a number of modifications to the C3 carboxylic acid were well tolerated. However, in the absence of the C6 ester group, similar modifications to the C3 carboxyl group caused loss of activity. Selected compounds were evaluated for their ability to inhibit cholesterol biosynthesis in vivo in rats 1 and 6 h postadministration. Analogues of squalestatin 1 (S1) modified at C3 were found to possess a shorter duration of effect in vivo which is reflected in their substantially reduced ability to lower serum cholesterol levels in marmosets. Significant cholesterol lowering (up to 62%) for the C3 hydroxymethyl analogue 1b was observed only when this compound was dosed three times a day for 3 days.

The squalestatins: decarboxy and 4-deoxy analogues as potent squalene synthase inhibitors.

Squalestatins without either the hydroxy group at C-4 or the carboxylic acid at C-3 or C-4 were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase (SQS) in vitro. These modifications were well tolerated for compounds with the 4,6-dimethyloctenoate ester at C-6 (S1 series). However in analogues without the C-6 ester (H1 series), removal of the C-4 hydroxy group gave compounds with reduced potency, whereas decarboxylation at C-3 resulted in a dramatic loss of SQS inhibitory activity. In comparison with S1 1, C-4 deoxyS1 3 and C-3 decarboxyS1 10 have shorter in vivo durations of action on the inhibition of hepatic cholesterol biosynthesis in rats. C-4 deoxyS1 3 retains good serum cholesterol-lowering ability in marmosets, while C-3 decarboxyS1 10 showed only a marginal effect even at high dose.

Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids.

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [3H]mevalonate into ubiquinone-9 and -10 increases 3-4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.

The squalestatins: novel inhibitors of squalene synthase. Enzyme inhibitory activities and in vivo evaluation of C1-modified analogues.

Squalestatin analogues modified in the C1 side chain were prepared and evaluated for their ability to inhibit rat liver microsomal and Candida squalene synthase (SQS) in vitro. While maintaining the 4,6-dimethyloctenoate or 4,6-dimethyloctanoate ester groups at C6, a number of modifications to the C1 side chain were well tolerated. However, in the absence of the C6 ester group, similar modifications to the C1 side chain caused substantial loss of activity. Compounds were also evaluated for their ability to inhibit cholesterol biosynthesis in vivo in rats and to reduce serum cholesterol levels in marmosets. These studies revealed that compounds with similar SQS inhibitory activities can possess different in vivo durations of action and lipid-lowering abilities.

Squalestatin 1, a potent inhibitor of squalene synthase, which lowers serum cholesterol in vivo.

Squalestatin 1 is a member of a novel family of fermentation products isolated from a previously unknown Phoma species (Coelomycetes). Squalestatin 1 is a potent, selective inhibitor of squalene synthase, a key enzyme in cholesterol biosynthesis; in vitro, 50% inhibition of enzyme activity is observed at a concentration of 12 +/- 5 nM (range of 4-22 nM). Squalestatin 1 inhibits cholesterol biosynthesis from [14C]acetate by isolated rat hepatocytes (50% inhibition at 39 nM) and by rat liver in vivo. In marmosets, a species with a lipoprotein profile similar to that of man, squalestatin 1 lowers serum cholesterol by up to 75%. This compound will allow further investigation of the control of the sterol biosynthesis pathway and could also lead to the development of new therapies for elevated serum cholesterol.

Esterification and absorption of cholesterol: in vitro and in vivo observations in the rat.

Activity of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) in isolated rat enterocytes was reduced by approx. 75% following a single oral dose of Sandoz compound 58-035 (30 mg.kg-1). Despite this, the formation of [14C]cholesteryl esters from [1-14C]oleic acid remained unaffected in ACAT-inhibited cell preparations. The increase in serum cholesterol concentrations observed after overnight cholesterol/cholic acid (1%/0.5%) feeding to rats was abolished by pre-treatment with Sandoz compound 58-035 (30 mg.kg-1). These results can be reconciled with a previously proposed model for the transmembrane movement of cholesterol which implicates ACAT-independent esterification and hydrolysis as a transport mechanism for the movement of cholesterol across the enterocyte apical membrane.

Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations.

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.

Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase.

Two thermolytic peptides containing the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase have been isolated and sequenched by Edman degradation and fast atom bombardment mass spectrometry. The sequence (V-A-G-Y-S-Y-G) contains the motif G-X-S-X-G found around the reactive serine residue of all known serine proteinases and esterases.

Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation.

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.