Sianna Panagiotopoulos

Low testosterone levels are common and associated with insulin resistance in men with diabetes.

CONTEXT Low testosterone levels are common in men with type 2 diabetes and may be associated with insulin resistance. OBJECTIVE We investigated prevalence of testosterone deficiency and the relationship between testosterone and insulin resistance in a large cohort of men with type 2 and type 1 diabetes. DESIGN The study was a cross-sectional survey of 580 men with type 2 diabetes and 69 men with type 1 diabetes. A subgroup of 262 men with type 2 diabetes was then reassessed after a median of 6 months. RESULTS Forty-three percent of men with type 2 diabetes had a reduced total testosterone, and 57% had a reduced calculated free testosterone. Only 7% of men with type 1 diabetes had low total testosterone. By contrast, 20.3% of men with type 1 diabetes had low calculated free testosterone, similar to that observed in type 2 diabetes (age-body mass index adjusted odds ratio = 1.4; 95% confidence interval = 0.7-2.9). Low testosterone levels were independently associated with insulin resistance in men with type 1 diabetes as well as type 2 diabetes. Serial measurements also revealed an inverse relationship between changes in testosterone levels and insulin resistance. CONCLUSIONS Testosterone deficiency is common in men with diabetes, regardless of the type. Testosterone levels are partly influenced by insulin resistance, which may represent an important avenue for intervention, whereas the utility of testosterone replacement remains to be established in prospective trials.

Modulation of Soluble Receptor for Advanced Glycation End Products by Angiotensin-Converting Enzyme-1 Inhibition in Diabetic Nephropathy

Recent studies have identified that first-line renoprotective agents that interrupt the renin-angiotensin system not only reduce BP but also can attenuate advanced glycation end product (AGE) accumulation. This study used in vitro, preclinical, and human approaches to explore the potential effects of these agents on the modulation of the receptor for AGE (RAGE). Bovine aortic endothelial cells that were exposed to the angiotensin-converting enzyme inhibitor (ACEi) ramiprilat in the presence of high glucose demonstrated a significant increase in soluble RAGE (sRAGE) secreted into the medium. In streptozotocin-induced diabetic rats, ramipril treatment (ACEi) at 3 mg/L for 24 wk reduced the accumulation of skin collagen-linked carboxymethyllysine and pentosidine, as well as circulating and renal AGE. Renal gene upregulation of total RAGE (all three splice variants) was observed in ACEi-treated animals. There was a specific increase in the gene expression of the splice variant C-truncated RAGE (sRAGE). There were also increases in sRAGE protein identified within renal cells with ACEi treatment, which showed AGE-binding ability. This was associated with decreases in renal full-length RAGE protein from ACEi-treated rats. Decreases in plasma soluble RAGE that were significantly increased by ACEi treatment were also identified in diabetic rats. Similarly, there was a significant increase in plasma sRAGE in patients who had type 1 diabetes and were treated with the ACEi perindopril. Complexes between sRAGE and carboxymethyllysine were identified in human and rodent diabetic plasma. It is postulated that ACE inhibition reduces the accumulation of AGE in diabetes partly by increasing the production and secretion of sRAGE into plasma.

Angiotensin converting enzyme inhibition reduces retinal overexpression of vascular endothelial growth factor and hyperpermeability in experimental diabetes.

Aims/hypothesis. Angiotensin converting enzyme (ACE) inhibition has been recently suggested to have retinoprotective actions in diabetic patients but the mechanism of this effect is not known. In vitro, angiotensin II stimulates expression of vascular endothelial growth factor (VEGF), a permeability-inducing and endothelial cell specific angiogenic factor which has been implicated in the pathogenesis of diabetic retinopathy in humans and in experimental animals. We sought to determine the effects of ACE inhibition on retinal VEGF expression and permeability in experimental diabetic retinopathy.¶Methods. Streptozotocin-induced diabetic rats and control animals were assigned at random to receive ACE inhibitor treatment or vehicle. At 24 weeks the retinal VEGF protein gene expression was assessed by northern blot analysis and in situ hybridisation. Retinal permeability to albumin was measured using a double isotope technique.¶Results. Experimental diabetes was associated with cell specific two to fourfold increase in retinal VEGF protein gene expression (p < 0.01) and a 2-fold increase in retinal vascular permeability to albumin (p < 0.01). The localization of VEGF expression in the retina was not altered in animals with experimental diabetes. Angiotensin converting enzyme inhibitor treatment of diabetic rats reduced diabetes-associated changes in VEGF gene expression and vascular permeability.¶Conclusion/interpretation. These findings implicate the renin-angiotensin system in the VEGF overexpression and hyperpermeability which accompany diabetic retinopathy and provide a potential mechanism for the beneficial effects of ACE inhibition in diabetic retinal disease. [Diabetologia (2000) 43: 1360–1367]

Urinary Proteomics for Early Diagnosis in Diabetic Nephropathy

Diabetic nephropathy (DN) is a progressive kidney disease, a well-known complication of long-standing diabetes. DN is the most frequent reason for dialysis in many Western countries. Early detection may enable development of specific drugs and early initiation of therapy, thereby postponing/preventing the need for renal replacement therapy. We evaluated urinary proteome analysis as a tool for prediction of DN. Capillary electrophoresis–coupled mass spectrometry was used to profile the low–molecular weight proteome in urine. We examined urine samples from a longitudinal cohort of type 1 and 2 diabetic patients (n = 35) using a previously generated chronic kidney disease (CKD) biomarker classifier to assess peptides of collected urines for signs of DN. The application of this classifier to samples of normoalbuminuric subjects up to 5 years prior to development of macroalbuminuria enabled early detection of subsequent progression to macroalbuminuria (area under the curve [AUC] 0.93) compared with urinary albumin routinely used to determine the diagnosis (AUC 0.67). Statistical analysis of each urinary CKD biomarker depicted its regulation with respect to diagnosis of DN over time. Collagen fragments were prominent biomarkers 3–5 years before onset of macroalbuminuria. Before albumin excretion starts to increase, there is a decrease in collagen fragments. Urinary proteomics enables noninvasive assessment of DN risk at an early stage via determination of specific collagen fragments.

Renoprotective effects of a novel inhibitor of advanced glycation.

Aims/hypothesis. ALT-946, an inhibitor of advanced glycation with a minimal inhibitory effect on nitric oxide synthase, was compared with aminoguanidine in experimental diabetic nephropathy.¶Methods. In vitro and in vivo assays were used to assess the ability of ALT-946 to inhibit AGE-protein cross-link formation. Diabetic animals were randomly allocated into groups receiving aminoguanidine for 32 weeks, ALT-946 or vehicle (untreated). As a delayed intervention protocol, an additional diabetic group was treated with ALT-946 from week 16 to week 32 of the study. Non-diabetic rats were studied concurrently. Systolic blood pressure, body weight, plasma glucose, glycated haemoglobin and urinary albumin excretion were measured serially. Accumulation of advanced-glycation end products in the kidney was assessed by immunohistochemistry.¶Results. The ALT-946 inhibitor was more potent than aminoguanidine in inhibiting AGE-protein cross-linking both in vitro and in vivo. Increased albuminuria observed in diabetic rats was attenuated in all three treatment groups. We found no difference in body weight, blood pressure or glycaemic control with any of the treatments. The untreated diabetic group had a twofold increase in glomerular staining for advanced-glycation end products compared with the diabetic groups which received treatment.¶Conclusion/interpretation. ALT-946 is a potent inhibitor of advanced renal glycation end-product accumulation and reproduces the renoprotective effects of aminoguanidine. Therefore, ALT-946 should be considered as a treatment for preventing or retarding diabetic nephropathy. [Diabetologia (2001) 44: 108–114]

Progressive Decline in Renal Function in Diabetic Patients With and Without Albuminuria

This study describes patterns of progression of albuminuria and renal function in a subgroup of 40 patients from a total cohort of 211 diabetic patients (118 type I, 93 type II) followed over a period of 8–14 years. Forty patients (18 with type I diabetes, 22 with type II diabetes) showed progressive increases in albumin excretion rate (AER) and/or decreases in creatinine clearance (CC) during the study period. Of these, AER alone increased in 15 patients, AER increased and CC decreased in 13 patients, and CC alone decreased in 12 patients, with a similar distribution of type I and type II diabetic patients in each group. Of the 28 patients who showed an increase in albuminuria, AER increased at an annual rate of 30–40%, resulting in a 4- to 8-fold increase in AER to > 20 μg/min during the study. Of the 25 patients who showed a decrease in renal function, CC decreased at an annual rate of 4–5 ml/min, resulting in an approximate halving of CC to < 90 ml/min during the study. The rate of fall in CC was not related to the presence or absence of concomitant increases in albuminuria. However, a significant preponderance of women in the group showed a decline in CC alone. The decline in CC was associated with an increase in plasma creatinine as well as a progressive decrease in urinary creatinine excretion, but the underlying mechanisms remain unexplained. These data support the concept that a subgroup of diabetic patients may show a decline in renal function in the absence of significant increases in AER. Additional functional and structural data will be needed to determine if these patients have diabetic nephropathy. However, this study does suggest that albuminuria alone may not predict renal functional changes in all diabetic patients.

The clinical significance of hyperfiltration in diabetes

Glomerular filtration rate is commonly elevated in early diabetes and patients with this symptom are arbitrarily considered to have hyperfiltration. The prevalence of hyperfiltration in type 1 diabetes varies from less than 25% to more than 75%. The corresponding figures in type 2 diabetes are significantly lower, ranging between 0% and more than 40%. Several factors, methodological and biological, may contribute to the wide variation in estimates of hyperfiltration prevalence. Methodological differences in measurement and evaluation of GFR apply in particular to the handling of plasma disappearance curves of filtration markers. Biological factors that may influence GFR in the hyperfiltration range include glycaemic control, diabetes duration, BMI, sex, pubertal status in type 1 diabetes and age in type 2 diabetes. Hyperglycaemia may influence GFR and albuminuria, and may therefore confound the evaluation of hyperfiltration as an independent risk factor for diabetic nephropathy. Adequate assessment of the relationship between glycaemic control, GFR and AER therefore requires serial measurements of all three variables followed by multivariate analysis. A recent meta-analysis of ten type 1 diabetes studies concluded that the presence of hyperfiltration at baseline more than doubled the risk of developing micro- or macroalbuminuria at follow-up. However, not all studies allowed for confounding factors or regression dilution bias. Future studies will therefore need to address the independent role of hyperfiltration, not only in the evolution of albuminuria, but also in the subsequent decline of GFR.

Interactions between Angiotensin II and NF-κB–Dependent Pathways in Modulating Macrophage Infiltration in Experimental Diabetic Nephropathy

NF-kappaB-dependent pathways play an important role in macrophage infiltration and kidney injury. NF-kappaB is regulated by angiotensin II (AII). However, the role of this pathway in diabetic nephropathy has not been clearly delineated. First, the activation of NF-kappaB, monocyte chemoattractant protein-1 (MCP-1), and macrophage infiltration in the diabetic kidney were explored, in a temporal manner. The active subunit of NF-kappaB, p65, was elevated in the diabetic animals in association with increased MCP-1 gene expression and macrophage infiltration. Second, the effects of treatment for 4 wk with the AII type 1 receptor antagonist valsartan, the AII type 2 receptor antagonist PD123319, or pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB and on these parameters were assessed. These treatments were associated with a reduction in p65 activation, MCP-1 gene expression, and macrophage infiltration. These findings demonstrate a role for activation of NF-kappaB, in particular the p65 subunit, in the pathogenesis of early renal macrophage infiltration in experimental diabetes. In the context of the known proinflammatory effects of AII, it is postulated that the renoprotection conferred by angiotensin II receptor antagonism is at least partly related to the inhibition of NF-kappaB-dependent pathways.

Calcium-mediated damage during post-ischaemic reperfusion

Ca2+ gain during reperfusion after an ischaemic episode may be of primary importance in the development of cell death and tissue necrosis. These studies were undertaken to examine the possibility of modifying Ca2+ gain by introducing interventions only upon reperfusion. Electron microscopy did not reveal the presence of holes in the sarcolemma prior to reperfusion, indicating that Ca2+ entry at the moment of reperfusion is not through rents in the sarcolemma. Reperfusion with acidotic buffer (pH 6.4 or 6.6) after 60 min ischaemia attenuated Ca2+ gain. However, this attenuation persisted only as long as the acidotic conditions were maintained. Conversely, reperfusion under alkalotic conditions (pH 7.9) exacerbated Ca2+ gain. Reperfusion with hypoxic buffer after 60 min ischaemia did not alter Ca2+ gain, but readmission of oxygen after a period of hypoxic post-ischaemic reperfusion triggered a further gain in Ca2+. Addition of 5 microM nifedipine to the reperfusion medium slowed Ca2+ gain, while 1 microM Bay K 8644, a calcium agonist, enhanced the gain at early times of reperfusion. Addition of 20 microM W-7, a calmodulin antagonist, to the reperfusion medium had no effect on post-ischaemic Ca2+ gain. Therefore, we have shown that it is possible to alter post-ischaemic Ca2+ gain by introducing interventions only upon reperfusion. However, Ca2+ gain could only be delayed, rather than prevented and ultimately no long-term protection was achieved. These results indicate that post-ischaemic Ca2+ gain is highly pH-sensitive and that entry of Ca2+ through voltage-activated slow channels may contribute to the early gain.

Relative contributions of advanced glycation and nitric oxide synthase inhibition to aminoguanidine-mediated renoprotection in diabetic rats.

Summary Advanced glycation end products (AGEs) have previously been shown to be increased in the diabetic kidney. Aminoguanidine, an inhibitor of advanced glycation, has been shown to attenuate the development of AGEs as well as the progression of renal disease in experimental diabetes. However, the precise mechanisms through which aminoguanidine acts remain to be elucidated since it is also able to act as an inhibitor of nitric oxide synthase (NOS). This study has therefore compared the effects of aminoguanidine with the effects of two other inhibitors of NOS, L -NAME and methylguanidine, on the development of experimental diabetic nephropathy. Diabetic rats were randomised to receive no treatment, aminoguanidine (1 g/l in drinking water), L -NAME (5 mg/l in drinking water) or methylguanidine (1 g/l in drinking water). Diabetic rats had increased levels of albuminuria and urinary nitrite/nitrate excretion when compared to control rats. Renal AGEs measured by fluorescence as well as by a carboxymethyllysine reactive radioimmunoassay, were elevated in diabetic rats. No changes in inducible NOS (iNOS) protein expression were detected in experimental diabetes nor did aminoguanidine affect iNOS expression. Aminoguanidine did not affect blood glucose or HbA1c but it did prevent increases in albuminuria, urinary nitrites/nitrates and renal AGE levels as measured by fluorescence and radioimmunoassay. L -NAME and methylguanidine did not retard the development of albuminuria, nor did they prevent increases in renal AGE levels, as assessed by fluorescence. However, these treatments did prevent increases in AGEs, as measured by radioimmunoassay. This study indicates that the renoprotective effect of aminoguanidine in experimental diabetes cannot be reproduced by L -NAME or methylguanidine. It is likely that the effect of aminoguanidine is mediated predominantly by decreased AGE formation rather than via NOS inhibition. It also raises the possibility that inhibition of fluorescent AGE formation may be more renoprotective than inhibition of the formation of carboxymethyllysine-containing AGEs. [Diabetologia (1997) 40: 1141–1151]