Correlates of muscle strength in diabetes: The study on the assessment of determinants of muscle and bone strength abnormalities in diabetes (SAMBA).

BACKGROUND AND AIMS: Apart from late motor nerve dysfunction, factors affecting muscle strength in diabetes are largely unknown. This study was aimed at assessing muscle strength correlates in diabetic subjects encompassing a wide range of peripheral nerve function and various degrees of micro and macrovascular complications. METHODS AND RESULTS: Four-hundred consecutive patients with type 1 and 2 diabetes (aged 46.4 ± 13.9 and 65.8 ± 10.3 years, respectively) from the Study on the Assessment of Determinants of Muscle and Bone Strength Abnormalities in Diabetes (SAMBA) were examined for upper and lower body muscle isometric maximal voluntary contraction by dynamometry. Univariate and multivariate regression analyses were applied to identify strength correlates. Isometric force at both the upper and lower limbs was significantly lower in subjects with than in those without any complication. At univariate analysis, it was strongly associated with age, diabetes duration, physical activity (PA) level, cardio-respiratory fitness, anthropometric parameters, surrogate measures of complications, and parameters of sensory and autonomic, but not motor (except amplitude) neuropathy. Multivariate analysis revealed that upper and lower body strength correlated independently with male gender and, inversely, with age, autonomic neuropathy score (or individual autonomic function abnormalities), and vibration perception threshold, but not sensory-motor neuropathy score. Diabetes duration and PA level were excluded from the model. CONCLUSIONS: Both upper and lower body muscle strength correlate with measures of diabetic complications and particularly with parameters of sensory and especially autonomic nerve function, independently of diabetes duration and PA level, thus suggesting the involvement of mechanisms other than manifest motor nerve impairment.

Relationship of exercise volume to improvements of quality of life with supervised exercise training in patients with type 2 diabetes in a randomised controlled trial: the Italian Diabetes and Exercise Study (IDES).

AIMS/HYPOTHESIS: A positive impact of exercise intervention programmes on quality of life (QoL) may be important for long-term patient compliance to exercise recommendations. We have previously shown that QoL improves significantly with supervised exercise, whereas it worsens with counselling alone, in patients with type 2 diabetes from the Italian Diabetes and Exercise Study (IDES). Here, we report data on the relationship between changes in QoL and volume of physical activity/exercise in these individuals. METHODS: This multicentre parallel randomised controlled, open-label, trial enrolled sedentary patients with type 2 diabetes (n = 606 of 691 eligible) in 22 outpatient diabetes clinics. Patients were randomised by centre, age and diabetes treatment using a permuted-block design to twice-a-week supervised aerobic and resistance training plus exercise counselling (exercise group) versus counselling alone (control group) for 12 months. Health-related QoL was assessed by the 36-Item Short Form (SF-36) Health Survey. RESULTS: In the exercise group (n = 268 of 303 randomised), there was a trend for increasing QoL with increasing exercise volume, with significant improvement of the physical component summary (PCS) measure only above 17.5 metabolic equivalents h⁻¹ week⁻¹ and a clear volume-relationship for the mental component summary (MCS) measure. A relationship with volume of physical activity also was observed in the control group (n = 260 of 303 randomised), despite overall deterioration of all scores. Independent correlates of improvements in both PCS and MCS were exercise volume, study arm and, inversely, baseline score. CONCLUSIONS/INTERPRETATION: This large trial shows a relationship between changes in physical and mental health-related QoL measures and volume of physical activity/exercise, with supervised exercise training also providing volume-independent benefits.

Glomerular barrier dysfunction in glomerulosclerosis- resistant Milan rats with experimental diabetes: the role of renal haemodynamics.

Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease. Genetically determined hypertrophy of intrarenal arteries has been suggested as the putative mechanism preventing transmission of systemic hypertension to the glomerular microcirculation or diabetes-induced loss of autoregulation, which lead to glomerular hypertension and consequent podocyte injury and proteinuria. This study aimed to investigate glomerular barrier function and structure in ageing and diabetic MHS rats under basal conditions and after injection of 2.5 g of bovine serum albumin (BSA) causing increased workload and possibly removing haemodynamic protection by inducing renal cortical vasodilatation. Genetically related rats of the Milan normotensive strain (MNS) served as a proteinuric counterpart. No change in renal function or structure was detected in diabetic MHS rats, whereas MNS rats developed diabetic nephropathy superimposed on that occurring spontaneously in this strain. Diabetic, but not non-diabetic, MHS rats showed significantly reduced synaptopodin and nephrin expression, though to a lesser extent than non-diabetic and diabetic MNS rats, together with unchanged podocyte number, density and structure and no proteinuria. Agrin expression was significantly altered in diabetic versus non-diabetic MHS animals, whereas collagen I was expressed only in diabetic MHS rats and collagen IV content did not change significantly between the two groups. Upon BSA injection, proteinuria increased markedly and abundant BSA was detected only in kidneys from diabetic MHS rats. BSA injection was associated with changes in intrarenal arteries suggesting vasodilatation, without any influx of inflammatory cells. These data indicate that while MNS rats show marked changes in the glomerular filtration barrier with either age or diabetes, glomerulosclerosis-resistant MHS rats develop only minor diabetes-induced podocyte (and extracellular matrix) alterations, which are not associated with proteinuria unless they are unmasked by an increased workload or removal of the haemodynamic protection.

Increased glomerular cell (podocyte) apoptosis in rats with streptozotocin-induced diabetes mellitus: role in the development of diabetic glomerular disease.

AIMS/HYPOTHESIS: Podocyte loss by apoptosis, in addition to favouring progression of established diabetic nephropathy, has been recently indicated as an early phenomenon triggering the initiation of glomerular lesions. This study aimed to assess the rate of glomerular cell death and its relationship with renal functional, structural and molecular changes in rats with experimental diabetes. METHODS: Male Sprague-Dawley rats with streptozotocin-induced diabetes and coeval non-diabetic control animals were killed at 7 days and at 2, 4 and 6 months for the assessment of apoptosis, renal function, renal structure and the expression of podocyte markers and apoptosis- and cell cycle-related proteins. RESULTS: Glomerular cell apoptosis was significantly increased in diabetic vs non-diabetic rats at 4 months and to an even greater extent at 6 months, with podocytes accounting for 70% of apoptosing cells. The increase in apoptosis was preceded by increases in proteinuria, albuminuria and mean glomerular and mesangial areas, and by reductions in glomerular cell density and content of synaptopodin and Wilms' tumour protein-1. It coincided with the development of mesangial expansion and glomerular sclerosis, and with the upregulation/activation both of tumour protein p53, which increased progressively throughout the study, and of p21 (also known as cyclin-dependent kinase inhibitor 1A, CIP1 and WAF1), which peaked at 4 months and decreased thereafter. CONCLUSIONS/INTERPRETATION: Glomerular cell (podocyte) apoptosis is not an early feature in the course of experimental diabetic glomerulopathy, since it is preceded by glomerular hypertrophy, which may decrease glomerular cell density to the point of inducing compensatory podocyte hypertrophy. This is associated with reduced podocyte protein expression (podocytopathy) and proteinuria, and ultimately results in apoptotic cell loss (podocytopenia), driving progression to mesangial expansion and glomerular sclerosis.

Ablation of the gene encoding p66Shc protects mice against AGE-induced glomerulopathy by preventing oxidant-dependent tissue injury and further AGE accumulation.

AIMS/HYPOTHESIS: AGEs have been implicated in renal disease associated with ageing, diabetes and other age-related disorders. Reactive oxygen species (ROS) promote formation of AGEs, which cause AGE-receptor-mediated ROS generation with activation of signalling pathways leading to tissue injury and further AGE accumulation. ROS generation is regulated by the Src homology 2 domain-containing transforming protein C1 (Shc1) isoform p66(Shc), whose deletion has been shown to protect from tissue injury induced by ageing, diabetes, hyperlipidaemia and ischaemia-reperfusion by preventing oxidative stress. This study was aimed at assessing the role of p66(Shc) in the modulation of oxidative stress and oxidant-dependent renal injury induced by AGEs. METHODS: For 10 weeks, male p66 (shc) knockout (KO) and wild-type (WT) mice were injected with 60 microg/day albumin modified or unmodified by N epsilon-(carboxymethyl) lysine (CML). Mice were then killed for the assessment of renal function and structure, as well as systemic and renal tissue oxidative stress. RESULTS: Upon CML injection, KO mice, in contrast to WT mice, showed no or only mild forms of proteinuria, glomerular hypertrophy, mesangial expansion, glomerular sclerosis, renal/glomerular cell apoptosis and extracellular matrix upregulation. Moreover, KO mice had lower circulating and tissue AGEs than WT mice and unchanged plasma isoprostane 8-epi-prostaglandin-F(2alpha) levels, renal/glomerular CML, 4-hydroxy-2-nonenal, AGE receptor and NAD(P)H oxidase 4 (NOX4) content (and expression of the corresponding genes), and nuclear factor kappaB activation (NFkappaB). Mesangial cells from KO mice exposed to CML showed no or slight increase in ROS levels and NFkappaB activation, again at variance with WT cells. CONCLUSIONS/INTERPRETATION: These data indicate that p66(Shc) participates in the pathogenesis of AGE-dependent glomerulopathy by mediating AGE-induced tissue injury and further AGE formation through ROS-dependent mechanisms involving NFkappaB activation and upregulation of Nox4 expression and NOX4 production.

Accelerated diabetic glomerulopathy in galectin-3/AGE receptor 3 knockout mice.

Several molecules were shown to bind advanced glycation end products (AGEs) in vitro, but it is not known whether they all serve as AGE receptors and which functional role they play in vivo. We investigated the role of galectin-3, a multifunctional lectin with (anti)adhesive and growth-regulating properties, as an AGE receptor and its contribution to the development of diabetic glomerular disease, using a knockout mouse model. Galectin-3 knockout mice obtained by gene ablation and the corresponding wild-type mice were rendered diabetic with streptozotocin and killed 4 months later, together with age-matched nondiabetic controls. Despite a comparable degree of metabolic derangement, galectin-3-deficient mice developed accelerated glomerulopathy vs. the wild-type animals, as evidenced by the more pronounced increase in proteinuria, extracellular matrix gene expression, and mesangial expansion. This was associated with a more marked renal/glomerular AGE accumulation, indicating it was attributable to the lack of galectin-3 AGE receptor function. The galectin-3-deficient genotype was associated with reduced expression of receptors implicated in AGE removal (macrophage scavenger receptor A and AGE-R1) and increased expression of those mediating cell activation (RAGE and AGE-R2). These results show that the galectin-3-regulated AGE receptor pathway is operating in vivo and protects toward AGE-induced tissue injury in contrast to that through RAGE.

Increased retinal endothelial cell monolayer permeability induced by the diabetic milieu: role of advanced non-enzymatic glycation and polyol pathway activation.

BACKGROUND: Increased vascular permeability could be involved in the pathogenesis of diabetic retinopathy. The present study was aimed at assessing whether high glucose concentrations can impair retinal endothelial cell barrier function directly, irrespective of changes in other determinants of permeability, and the role of non-enzymatic glycation and polyol pathway activation in these alterations. METHODS: Bovine retinal endothelial cells (BREC) were exposed for various periods to high glucose vs iso-osmolar mannitol and normal glucose containing media+/-agents mimicking or inhibiting advanced glycation end product (AGE) formation and polyol pathway activation. Monolayer permeability was assessed by measuring the transendothelial passage of (125)I-labeled proteins. RESULTS: Permeability increased significantly (up to +70%) in BREC exposed to high glucose, but not to mannitol, for 1-30 days, vs normal glucose control cells. Exposure to AGE-modified bovine serum albumin (BSA) (> or = 90%) and, to a lesser extent, sorbitol (+28%) mimicked the high glucose effect. The AGE formation and nitric oxide synthase (NOS) inhibitor aminoguanidine significantly reduced (by 60%) changes induced by 30-day exposure to high glucose, whereas methylguanidine, which inhibits only NOS activity, did not affect permeability. Aldose reductase or sorbitol dehydrogenase inhibitors decreased (by approximately 40%) the enhanced leakage produced by 1-day, but not 30-day, incubation in high glucose. CONCLUSIONS: The present results indicate that high glucose is capable of impairing retinal endothelial cell barrier function directly and that non-enzymatic glycation and polyol pathway activation may mediate these changes, with AGEs participating in the long-term alterations and increased flux through the sorbitol pathway in the short-term effect.

Role of galectin-3 as a receptor for advanced glycosylation end products.

The advanced glycosylation end product (AGE)-binding proteins identified so far include the components of the AGE-receptor complex p60, p90 and galectin-3, receptor for advanced glycosylation end products (RAGE), and the macrophage scavenger receptor types I and II. Galectin-3 interacts with beta-galactoside residues of several cell surface and matrix glycoproteins through the carbohydrate recognition domain and is also capable of peptide-peptide associations mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple functions, including the modulation of cell adhesion, the control of cell cycle, and the mRNA splicing activity. Moreover, in macrophages, astrocytes, and endothelial cells, galectin-3 has been shown to exhibit a high-affinity binding for AGEs; the lack of a transmembrane anchor sequence or signal peptide suggests that it associates with other AGE-receptor components rather than playing an independent role as AGE-receptor. In tissues that are targets of diabetic vascular complications, such as the mesangium and the endothelium, galectin-3 is not expressed or only weakly expressed under basal conditions, at variance with p90 and p60 but becomes detectable with aging and is induced or up-regulated by the diabetic milieu, which only slightly affects the expression of p90 or p60. This (over)expression of galectin-3 may in turn modulate AGE-receptor-mediated events by modifying the function of the AGE-receptor complex, which could play a role in the pathogenesis of target tissue injury. Up-regulated galectin-3 expression may also exert direct effects on tissue remodeling, independently of AGE ligands, by virtue of its adhesive and growth regulating properties.

The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression.

Nonenzymatic glycation has been implicated in the pathogenesis of the dysregulated tissue remodeling that characterizes diabetic glomerulopathy, via the formation of advanced glycation end products (AGEs) and their binding to cell surface receptors. Several AGE-binding proteins have been identified so far, including p60, p90, and the adhesive and growth-regulating lectin galectin-3 (Gal-3), the components of the so-called AGE-receptor complex. This study aimed to evaluate the mesangial expression of the AGE-receptor complex and its modulation by the diabetic milieu, both in vivo, in non-diabetic versus streptozotocin-induced diabetic rats, and in vitro, in mesangial cells exposed to either normal glucose (NG) levels (5.5 mmol/l), as compared with high glucose (HG) levels (30 mmol/l) and iso-osmolar mannitol (M), or to native bovine serum albumin (BSA), as compared with glycated BSA with AGE formation (BSA-AGE) and glycated BSA in which AGE formation was prevented by aminoguanidine (BSA-AM). In vivo, Gal-3 protein and mRNA were not detectable in glomeruli from nondiabetic rats until 12 months after initiating the study. On the contrary, in diabetic rats, Gal-3 expression was observed at 2 months of disease duration, and it increased thereafter. Both p60 and p90 immunoreactivities were observed at the glomerular level with slightly increased expression of p90, but not p60, in diabetic versus nondiabetic animals. In vitro, Gal-3 was not detectable in mesangial cells cultured in NG (although it became evident after a certain number of passages in culture), whereas Gal-3 was detectable in cells grown on BSA. Prolonged exposure (2-4 weeks) of mesangial cells to HG but not to M, as well as growing cells on BSA-AGE and, to a lesser extent, BSA-AM, induced or significantly increased the expression of Gal-3, both protein (up to 2.65-fold) and mRNA (up to 3.10-fold) and its secretion in the medium (by approximately 50%). Both p60 and p90 were demonstrated in mesangial cells under NG conditions, and the expression of p90, but not p60, was upregulated by approximately 20% by HG or BSA-AGE. These results indicate that 1) under basal conditions, Gal-3, unlike p90 and p60, is not detectable in the mesangium but becomes expressed with aging and 2) the diabetic milieu induces or upregulates Gal-3 production, whereas it increases only slightly the expression of p90, but not p60. Gal-3 expression or overexpression may modulate the AGE-receptor-mediated events by modifying the function of the AGE-receptor complex. Additionally, it may exert direct effects on tissue remodeling by virtue of its adhesive and growth-regulating properties.

Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb.

hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down-regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che-1, a novel human protein that interacts with hRPB11. Che-1 possesses a domain of high homology with Escherichia coli RNA polymerase final sigma-factor 70 and SV40 large T antigen. In addition, we report that Che-1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che-1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the trans-activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che-1 may be part of transcription regulatory complex.

Levels of expression of hRPB11, a core subassembly subunit of human RNA polymerase II, affect doxorubicin sensitivity and cellular differentiation.

We have previously shown that the human RNA polymerase II subunit 11 (hRPB11) is among the proteins specifically downregulated upon Doxorubicin (Dox) treatment of human cancer cell lines, and that Dox resistant clones derived upon drug selection express about 20% of the protein present in the original parental cell line. Given the prominent role that this subunit appears to have in eukaryotic cells, and the fact that its deletion causes lethality in yeast, we wanted to test the effect of the reintroduction of parental cell line levels of this subunit in Dox resistant colon cancer cells (LoVoDX). Stable transfectants of LoVoDX expressing parental (LoVoH) levels of hRPB11 showed a reduced sensitivity to the drug without changing the response of these cells to other chemotherapeutic agents, confirming a specific inverse correlation between cellular Dox sensitivity anti-hRPB11 levels of expression. In addition we show here that the levels of expression of this same RNA polymerase II subunit directly affect cellular differentiation, reducing the rate of cell proliferation, clonogenicity and increasing the expression of E-cadherin, a marker of epithelial cell differentiation. As expected from cells with these characteristics, upon in vivo administration of these clones in nude mice, we detected a significant reduction in the size and time of appearance of the primary tumors and overall metastatic capability. Finally, the role played by hRPB11 in regulating the transcription of specific genes is underlined by transient transfection experiments that show transactivation of the E-cadherin promoter by this protein.

Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity.

Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines. The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II. Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle. Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels. These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery.

Characterization of two novel YY1 binding sites in the polyomavirus late promoter.

NF-D is a ubiquitous nuclear factor that has been shown to bind specifically to a DNA element in the polyomavirus regulatory region. In this report, we demonstrate that NF-D is either identical or very similar to a transcription factor that has been variously named YY1, delta, NF-E1, UCRBP, or CF1. Moreover, we show the presence in the polyomavirus genome of a second DNA motif, located 40 bp from the first, which binds YY1/NF-D with high affinity. Both sites lie downstream of the major late transcription initiation sites. By site-directed mutagenesis, we demonstrate that both elements contribute positively to the activity of the late promoter, probably by a cooperative mechanism. We also demonstrate that the requirement of the YY1/NF-D function for late promoter activity varies with the cell line.

Thermal behavior of a human glioma cell line and its response to combinations of hyperthermia and lonidamine.

The effect of hyperthermia and lonidamine, alone and in combination, on the clonogenic activity of a human glioma cell line was investigated. The time-temperature relationship of asynchronous, exponentially growing cells was defined in the range of 40-45 degrees C. All survival curves were exponential and an Arrhenius plot for heat killing was linear over the temperature range tested, with an activation energy of 192 Kcal/mol. The survival curve of lonidamine-treated cells was also exponential after an initial shoulder. The analysis of the interaction between lonidamine and hyperthermia, performed by the isobolar method, demonstrated an additivity of response so that the effectiveness of the combined treatment was the result of two independent effects. Lonidamine inhibits the neoplastic growth mainly through an ATP depletion, but the thermal killing was not mediated by the drug-induced changes in the energy status of the cell. The effectiveness of the combined treatment was strongly influenced by the schedule of administration. In fact, the sequence lonidamine-->hyperthermia made the cells less sensitive to heat so that the pre-established end-point, i.e. 30% survival, was never achieved whichever combination was used. This "drug-induced heat resistance" was not associated with the induction of heat shock proteins, but rather with modification of cell cycle. On the contrary, showing a purely additive effect, the sequence hyperthermia-->lonidamine allowed achievement of the pre-established cell killing (70%), with exposure times (1-2 hr) and with a temperature (42 degrees C) generally accepted as clinically achievable. Therefore, also considering its low systemic toxicity, lonidamine may be useful in reducing the side effects of hyperthermia.

Survival response of a human glioma cell line to hyperthermia associated with rhein.

The effect of association of hyperthermia with the anti-inflammatory drug rhein (RH), 4,5-dihydroxyanthraquinone-2-carboxylic acid, on the clonogenic activity of human glioma cells has been examined. RH inhibits neoplastic growth mainly through an ATP depletion, but thermal cell killing is not mediated by the drug-induced changes in the energy status of the cell. The analysis of the interaction between RH and hyperthermia, performed with the isobolar method, demonstrates an additivity of the response so that the effectiveness of the combined treatment is the result of two independent effects. Although the effect of this combination is purely additive, RH allows us to achieve a pre-established cell killing with exposure times at 42 degrees C, which is generally accepted to be clinically achievable. RH might, therefore, be employed to reduce the side effects of hyperthermia without impairing its therapeutic effectiveness.

Protein recognition sites in polyomavirus enhancer: formation of a novel site for NF-1 factor in an enhancer mutant and characterization of a site in the enhancer D domain.

Polyomavirus mutants selected for modified host range exhibit DNA sequence alterations in the regulatory region, which consist mainly of duplications and/or deletions. Single base pair mutations have also been observed, which create or abolish DNA sequence motifs recognized by DNA-binding regulatory factors. The present work deals with the molecular characterization of a Polyoma mutant (PyNB11/1), selected for its high efficiency of growth in neuroblastoma cells. The enhancer region of PyNB11/1 displays a 91 bp tandem duplication harbouring a novel DNA sequence motif created at the boundary of the duplicated fragment. This motif is absent in the wild-type enhancer and is specifically recognized by a nuclear factor that belongs to the NF-1 family of transcription factors. We also report the characterization of an as yet unidentified DNA sequence motif in the D domain of the viral enhancer, that represents the binding site for a nuclear factor that is ubiquitous and comparably abundant in several murine cell types.