Milco Ciccarese

Reduction of Albumin Excretion Rate in Normotensive Microalbuminuric Type 2 Diabetic Patients During Long-Term Simvastatin Treatment

OBJECTIVE To study the long-term effects of simvastatin on urinary albumin excretion rate (AER) in normotensive microalbuminuric type 2 diabetic patients with hypercholesterolemia. RESEARCH DESIGN AND METHODS A total of 19 normotensive microalbuminuric hypercholesterolemic type 2 diabetic patients entered a double-blind crossover study for 2 years, receiving either simvastatin (20 mg/day) or placebo (each treatment for 1 year). RESULTS Simvastatin significantly decreased plasma cholesterol (total and LDL) after 52 weeks of treatment. A concomitant significant decrease of AER (25% from basal) with no significant changes in creatinine clearance was observed during the same period. CONCLUSIONS Our data are in keeping with the hypothesis that simvastatin might be used as an additional means to preserve renal function in microalbuminuric hypercholesterolemic type 2 diabetic patients.

Insulin sensitivity and glucose effectiveness: minimal model analysis of regular and insulin-modified FSIGT

The minimal model is widely used to evaluate insulin action on glucose disappearance from frequently sampled intravenous glucose tolerance tests (FSIGT). The common protocols are a regular (rFSIGT, single injection of 0.3 g/kg of glucose) and an insulin-modified test (mFSIGT, with an additional insulin administration at 20 min). This study compared the insulin sensitivity index (SI) and glucose effectiveness (SG) obtained in the same individual (16 normal subjects) with the two tests. SI was 7.11 ± 0.80 10-4 ⋅ min-1 ⋅ μU-1 ⋅ ml in rFSIGT and 6.96 ± 0.83 in mFSIGT ( P = 0.656), regression r = 0.92, P < 0.0001; SG was 0.0260 ± 0.0028 min-1 and 0.0357 ± 0.0052, respectively, statistically different ( P = 0.013) but still with a good regression ( r = 0.66, P = 0.0051). SG and insulin amount during the early period correlated ( r = 0.6, P = 0.015 in rFSIGT and r = 0.76, P = 0.0006 in mFSIGT). In summary, both FSIGTs with minimal model analysis provide the same SI, which is a very robust index. SG was different by 28% due probably to the relationship between SG and the amount of circulating insulin. In studies comparing groups, the simpler rFSIGT can still be used with the advantage of accounting for endogenous insulin, thus offering the possibility of direct inferences on the β-cell activity.The minimal model is widely used to evaluate insulin action on glucose disappearance from frequently sampled intravenous glucose tolerance tests (FSIGT). The common protocols are a regular (rFSIGT, single injection of 0.3 g/kg of glucose) and an insulin-modified test (mFSIGT, with an additional insulin administration at 20 min). This study compared the insulin sensitivity index (SI) and glucose effectiveness (SG) obtained in the same individual (16 normal subjects) with the two tests. SI was 7.11 +/- 0.80 10(-4).min-1.microU-1.ml in rFSIGT and 6.96 +/- 0.83 in mFSIGT (P = 0.656), regression r = 0.92, P < 0.0001; SG was 0.0260 +/- 0.0028 min-1 and 0.0357 +/- 0.0052, respectively, statistically different (P = 0.013) but still with a good regression (r = 0.66, P = 0.0051). SG and insulin amount during the early period correlated (r = 0.6, P = 0.015 in rFSIGT and r = 0.76, P = 0.0006 in mFSIGT). In summary, both FSIGTs with minimal model analysis provide the same SI, which is a very robust index. SG was different by 28% due probably to the relationship between SG and the amount of circulating insulin. In studies comparing groups, the simpler rFSIGT can still be used with the advantage of accounting for endogenous insulin, thus offering the possibility of direct inferences on the beta-cell activity.

Raised serum apolipoprotein (a) in active diabetic retinopathy

SummaryProgressive capillary occlusion often leads to severe retinopathy within 15–20 years of the onset of Type 1 (insulin-dependent) diabetes mellitus. Lipoprotein (a), a complex formed by apolipoprotein (a), apo B-100 and lipids, is considered an independent, genetically determined, predictor of cardiovascular disease. It may have antifibrinolytic properties in view of its similarity to plasminogen. To test the hypothesis that circulating lipoprotein (a) is associated with the process that leads to clinically active diabetic retinopathy, we measured the circulating levels of apolipoprotein (a) (which are strictly correlated with those of lipoprotein (a)) in two groups of patients with Type 1 diabetes of at least 15 years duration: 25 with active retinopathy and 27 without clinically detectable retinal lesions. Thirty-eight healthy subjects of the same age and sex served as controls. Serum apolipoprotein (a) was higher in the patients with active retinopathy (36(2-193) U/dl, geometric mean and range) than in those without clinically detectable retinal lesion (17(1–160)) and the control subjects (14(0–115)), p < 0.01 in both cases. The distribution of apolipoprotein (a) levels was skewed to the left, as expected, in the patients without clinically evident retinal lesions and the control groups, but there was a bimodal trend of distribution among those with active retinopathy. The levels of glycated haemoglobin were similar in the two groups of diabetic patients, and no significant differences were found for total and HDL cholesterol, triglycerides or apolipoproteins A1 and B between them and the control subjects. These preliminary results suggest that serum apolipoprotein (a) is elevated in patients with active retinopathy. The role of this lipoprotein as a predictor or a pathogenic effector of diabetic retinopathy, or both deserves further investigation.

Association of Trp64arg β3-adrenergic-receptor gene polymorphism with essential hypertension in the Sardinian population

OBJECTIVE To evaluate the possible association of three candidate gene polymorphisms with essential hypertension in the genetically homogeneous Sardinian population. SUBJECTS AND METHODS We studied 494 unrelated, nondiabetic subjects, 213 (43.2%) with essential hypertension. All subjects underwent a 75 g oral glucose tolerance test with determination of glycemia and insulinemia and serum lipids. The polymorphisms evaluated comprised Trp64Arg of the beta 3-adrenergic receptor, Gly40Ser of the glucagon receptor gene and the insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene. RESULTS Among the overall population studied, 48 (9.7%) were heterozygous carriers of the Trp64Arg polymorphism. The frequency of the Trp64Arg variant was significantly higher in hypertensives (13.6%) than normotensives (6.8%; chi 2 5.73, P = 0.017). The 48 subjects with the Trp64Arg variant had significantly higher (P < 0.049) serum triglyceride levels than the 446 with the Trp64Trp variant, while no significant differences were observed, either fasting or during the 75 g oral glucose tolerance test, in glycemia and insulinemia. No differences were found between hypertensive and normotensive subjects for ACE gene insertion/deletion polymorphism nor in the frequency of the Gly40Ser coding change in exon 2 of the glucagon receptor gene. CONCLUSIONS Our results are consistent with the thesis that the Trp64Arg polymorphism of the beta 3-adrenergic receptor gene is associated more often with the condition of high blood pressure than with normal blood pressure.

Physiological and genetic characterization of the Gly40Ser mutation in the glucagon receptor gene in the Sardinian population

Summary A Gly40Ser amino acid substitution in the glucagon receptor gene has been associated with non-insulin-dependent diabetes mellitus (NIDDM), but the questions raised about its physiological implications have not been resolved. The aim of this study was to determine the frequency of the Gly40Ser mutation in different regions of Sardinia and to investigate the physiological implications of the mutation in glucose and insulin homeostasis. We studied a population of 691 subjects, selected on the basis of their Sardinian origin. Only heterozygous subjects were found, 21 of 574 (3.6 %) in NIDDM patients and 5 of 117 in non-diabetic subjects (4.2 %). In northern Sardinia 3.4 % of the NIDDM patients were carriers of the Gly40Ser substitution, 1.4 % in central Sardinia, while 7.6 % carried the substitution in the Southern part. No significant differences were found between hypertensive and normotensive subjects with respect to the presence of Gly40Ser. Ten subjects with Gly40Ser were carefully matched for diabetic state, BMI, age, sex, and geographical origin with 10 patients with Gly40, and a glucagon infusion test was performed using 1, 3, 9 and 27 ng glucagon kg–1· min–1 for 30 min. Blood for determination of glucose, glucagon, and insulin concentrations was drawn at 15-min intervals from the Controlateral arm. Plasma glucagon increased dose-dependently during the infusion with no significant difference between the two groups. Carriers of Gly40Ser had a significantly lower (p < 0.02) increase in plasma glucose concentration in response to glucagon infusion compared to Gly40 homozygous subjects at all times, while the plasma insulin increase was not significantly different at any time. In conclusion, our results indicate that the Gly40Ser variation is not associated with NIDDM in the Sardinian population, and that its frequency varies in different parts of Sardinia. Moreover in vivo Gly40Ser plays a physiological role in the glucose homeostasis under glucagon control both in NIDDM and non-diabetic subjects. This latter result suggests that this amino acid substitution in the glucagon receptor may lead to a decreased blood glucose concentration because of the reduced stimulation of liver glucose output via the glucagon receptor. [Diabetologia (1997) 40: 89–94]

Proprotein Convertase Subtilisin Kexin Type 9 Inhibition for Autosomal Recessive Hypercholesterolemia - Brief Report

Objective—Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors lower low-density lipoprotein (LDL) cholesterol in the vast majority of patients with autosomal dominant familial hypercholesterolemia. Will PCSK9 inhibition with monoclonal antibodies, in particular alirocumab, be of therapeutic value for patients with autosomal recessive hypercholesterolemia (ARH)? Approach and Results—Primary lymphocytes were obtained from 28 genetically characterized ARH patients and 11 controls. ARH lymphocytes treated with mevastatin were incubated with increasing doses of recombinant PCSK9 with or without saturating concentrations of alirocumab. Cell surface LDL receptor expression measured by flow cytometry and confocal microscopy was higher in ARH than in control lymphocytes. PCSK9 significantly reduced LDL receptor expression in ARH lymphocytes albeit to a lower extent than in control lymphocytes (25% versus 76%, respectively), an effect reversed by alirocumab. Fluorescent LDL cellular uptake, also measured by flow cytometry, was reduced in ARH lymphocytes compared with control lymphocytes. PCSK9 significantly lowered LDL cellular uptake in ARH lymphocytes, on average by 18%, compared with a 46% reduction observed in control lymphocytes, an effect also reversed by alirocumab. Overall, the effects of recombinant PCSK9, and hence of alirocumab, on LDL receptor expression and function were significantly less pronounced in ARH than in control cells. Conclusions—PCSK9 inhibition with alirocumab on top of statin treatment has the potential to lower LDL cholesterol in some autosomal recessive hypercholesterolemia patients.

GAD65 and IA-2 autoantibodies are common in a subset of siblings of Sardinian Type 2 diabetes families

UNLABELLED Type 1 diabetes in Sardinia is very common in children, and we hypothesized that Latent Autoimmune Diabetes of Adult (LADA) might constitute a significant proportion of diabetes in adult Sardinian subjects. Since Type 2 diabetes is a familial disorder, we tested this hypothesis by investigating the prevalence of GAD65 and IA-2 autoantibodies (Ab) in Type 2 diabetes multiplex families of Sardinian ancestry enrolled in the Study Group for the Genetics of Diabetes in Sardinia. METHODS A total of 684 individuals were ascertained from 252 Sardinian Type 2 diabetes multiplex families with 2.4 affected siblings per family comprising 190 families with two affected, 37 with three, 15 with four, 7 with five, and 3 with six, in addition to 80 unaffected siblings. Controls were household contacts representing 204 healthy spouses of affected siblings. Diagnosis was at 35-69 years of age and insulin was not given in the first 4 years after diagnosis. GAD65Ab and IA-2Ab were determined in standard radioligand binding assays. RESULTS Among affected siblings GAD65Ab were positive in 8.8% of insulin-treated (n = 137; P = 0.0006), in 2.5% of non-insulin-treated (n = 467), and in 1.2% of non-diabetic siblings (n = 80) compared with 0.5% of controls (n = 204). IA-2Ab was positive in 6.6% insulin-treated (P = 0.04), 2.1% non-insulin-treated, and 2.5% non-diabetic siblings compared with 1.5% of controls. CONCLUSION A high frequency of GAD65Ab and IA-2Ab as markers of Type 1 diabetes was found among Type 2 diabetes siblings from Sardinian multiplex families despite excluding those who had been treated with insulin during the first 4 years of disease. Our data support the hypothesis that LADA may be common in Sardinian Type 2 diabetes and stress the importance of investigating markers of Type 1 diabetes in studies of Type 2 diabetes.

De Novo Everolimus-Based Therapy in Renal Transplant Recipients: Effect on Proteinuria and Renal Prognosis

BACKGROUND In large-scale clinical trials, the proliferation signal inhibitor (PSI) everolimus (EVL) combined with cyclosporine (CsA) and steroids, has been shown to be efficacious among de novo renal transplant recipients. Development of proteinuria has been shown to be an important predictor of renal dysfunction after conversion from CsA to a PSI-based regimen, and a key marker of allograft disease progression. Whether EVL de novo treatment is associated with a similar proteinuric effect is still under investigation. METHODS We compared the development of proteinuria among a cohort of 24 renal transplant recipients who were prescribed EVL (3 mg/d; n = 12; high-dose group) or 1.5 mg/d (n = 12; standard-dose group), in association with CsA, versus third control cohort of 12 patients who received mycophenolate mofetil (control group). EVL doses were adjusted to achieve trough blood levels of 3-8 ng/mL and 8-12 ng/mL among the standard and high-dose groups, respectively. We assessed renal function and protein excretion over a 2-year observation. RESULTS The high-dose group showed a trend toward greater proteinuria than the standard-dose on control groups. They showed significantly greater proteinuria from 9 months until 2 years; 0.86 +/- 0.5, 0.5 +/- 0.3, 0.47 +/- 0.2 g/24 h (P = .03 and P = .02, respectively, at 24 months). Mean proteinuria significantly correlated with mean EVL doses (n = .73; P = .0001). Concomitantly, the estimated glomerular filtration rate (eGFR) was significantly lower among patients treated with EVL 3.0 versus 1.5 mg/d (53.7 +/- 24 vs 73.04 +/- 17.6 mL/min; P = .037). Among patients in the standard-dose, the eGFR was consistently higher than the control group (62.6 +/- 29 mL/min). CONCLUSION EVL/CsA therapy is a safe alternative regimen for de novo renal transplant recipients. Higher EVL doses are correlated with greater increases in proteinuria. The standard EVL dose seems to be useful treatment strategy to prevent acute rejection episodes, with a better renal prognosis in the long term.

Proprotein Convertase Subtilisin Kexin Type 9 Inhibition for Autosomal Recessive Hypercholesterolemia

Objective—Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors lower low-density lipoprotein (LDL) cholesterol in the vast majority of patients with autosomal dominant familial hypercholesterolemia. Will PCSK9 inhibition with monoclonal antibodies, in particular alirocumab, be of therapeutic value for patients with autosomal recessive hypercholesterolemia (ARH)? Approach and Results—Primary lymphocytes were obtained from 28 genetically characterized ARH patients and 11 controls. ARH lymphocytes treated with mevastatin were incubated with increasing doses of recombinant PCSK9 with or without saturating concentrations of alirocumab. Cell surface LDL receptor expression measured by flow cytometry and confocal microscopy was higher in ARH than in control lymphocytes. PCSK9 significantly reduced LDL receptor expression in ARH lymphocytes albeit to a lower extent than in control lymphocytes (25% versus 76%, respectively), an effect reversed by alirocumab. Fluorescent LDL cellular uptake, also measured by flow cytometry, was reduced in ARH lymphocytes compared with control lymphocytes. PCSK9 significantly lowered LDL cellular uptake in ARH lymphocytes, on average by 18%, compared with a 46% reduction observed in control lymphocytes, an effect also reversed by alirocumab. Overall, the effects of recombinant PCSK9, and hence of alirocumab, on LDL receptor expression and function were significantly less pronounced in ARH than in control cells. Conclusions—PCSK9 inhibition with alirocumab on top of statin treatment has the potential to lower LDL cholesterol in some autosomal recessive hypercholesterolemia patients.

Kidney post-transplant monitoring of urinary glycosaminoglycans/proteoglycans and monokine induced by IFN-γ (MIG)

Allograft rejection during the first year after renal transplantation can lead to persistent allograft dysfunction and reduced long-term graft survival. Thus, it is important to define early predictors of kidney damage, less invasive than allograft biopsy. Urinary glycosaminoglycan/proteoglycan concentration and distribution, N-acetyl-β-(d)-glucosaminidase (NAG), and monokine induced by IFN-γ (MIG) levels were evaluated in the immediate post-transplant and during a 1-year follow-up. We observed increased urinary levels of MIG, urinary trypsin inhibitor and its degradation products, the lack of urinary heparan sulfate excretion, and the decreased chondroitin sulfate relative content at day 1 post-transplant in most patients who developed complications in the postoperative period. Moreover, urinary MIG levels showed significant correlations with NAG, C-reactive protein, and GFR at day 1 post-transplant. The monitoring of glycosaminoglycan/proteoglycan urinary pattern and the levels of urine MIG could serve as useful markers for predicting possible complications of transplantation, unraveling an early inflammatory state, on whose basis the immunosuppressive therapy could be appropriately modified.