A. Passaro

Plasma homocysteine, methylenetetrahydrofolate reductase mutation and carotid damage in elderly healthy women

Plasma homocysteine (Hcy) is an independent vascular risk factor. Its remethylation to methionine is regulated by the activity of the enzyme 5,10-methylene tetrahydrofolate reductase (MTHFR). A C-to-T substitution at nucleotide 677 of the MTHFR gene is frequently associated to hyperhomocysteinemia. In this study, we evaluated the relationship among MTHFR C677T polymorphism, Hcy and some ultrasonographic parameters at the level of carotid arteries in 120 elderly women with normal ECG, normal blood pressure values, total cholesterol <250 mg/dl, normal glucose tolerance, normal albumin excretion rate. In all subjects, we measured Hcy by HPLC, MTHFR mutation by polymerase chain reaction followed by HinfI digestion and intima-media thickness (IMT), peak velocity of the systolic flow (SP(V)), end-diastolic velocity (ED(V)) and resistance and pulsatility indexes of intracranial circulation (RI and PI) by ultrasound imaging. Twenty-eight women were homozygotes for the wild type allele (Ala/Ala), 72 were heterozygotes (Ala/Val) and 20 were homozygotes for the mutation (Val/Val). Groups were comparable for age, blood pressure values and plasma lipid levels. Hcy was higher in Val/Val group; moreover, after adjustment for confounding factors, Val/Val had significantly greater IMT and ED(V) (P<0.001 and P<0.05, respectively). Logistic analysis revealed that Val/Val genotype was the strongest risk factor for IMT (OR 30.8, 95% CI 2.82-335.6). Our results show that, in elderly healthy women, Val/Val homozygosity for C677T mutation in MTHFR gene could identify subjects at risk for asymptomatic carotid atherosclerotic impairment.

Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR

BackgroundBreast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15–3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique.ResultsWe investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis.ConclusionsThe study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs.

PPARγ Pro12Ala and ACE ID polymorphisms are associated with BMI and fat distribution, but not metabolic syndrome

BackgroundMetabolic Syndrome (MetS) results from the combined effect of environmental and genetic factors. We investigated the possible association of peroxisome proliferator-activated receptor-γ2 (PPARγ2) Pro12Ala and Angiotensin Converting Enzyme (ACE) I/D polymorphisms with MetS and interaction between these genetic variants.MethodsThree hundred sixty four unrelated Caucasian subjects were enrolled. Waist circumference, blood pressure, and body mass index (BMI) were recorded. Body composition was estimated by impedance analysis; MetS was diagnosed by the NCEP-ATPIII criteria. A fasting blood sample was obtained for glucose, insulin, lipid profile determination, and DNA isolation for genotyping.ResultsThe prevalence of MetS did not differ across PPARγ2 or ACE polymorphisms. Carriers of PPARγ2 Ala allele had higher BMI and fat-mass but lower systolic blood pressure compared with Pro/Pro homozygotes. A significant PPARγ2 gene-gender interaction was observed in the modulation of BMI, fat mass, and blood pressure, with significant associations found in women only. A PPARγ2-ACE risk genotype combination for BMI and fat mass was found, with ACE DD/PPARγ2 Ala subjects having a higher BMI (p = 0.002) and Fat Mass (p = 0.002). Pro12Ala was independently associated with waist circumference independent of BMI and gender.ConclusionsCarriers of PPARγ2 Ala allele had higher BMI and fat-mass but not a worse metabolic profile, possibly because of a more favorable adipose tissue distribution. A gene interaction exists between Pro12Ala and ACE I/D on BMI and fat mass. Further studies are needed to assess the contribution of Pro12Ala polymorphism in adiposity distribution.

Defective P2Y purinergic receptor function: a possible novel mechanism for impaired glucose transport

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT‐PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2‐deoxy‐D‐[1‐3H]glucose (2‐DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X‐mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co‐incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two‐fold larger amount of ATP compared to controls. Pre‐treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y‐dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes. J. Cell. Physiol. 197: 435–444, 2003© 2003 Wiley‐Liss, Inc.

A Defect in Glycogen Synthesis Characterizes Insulin Resistance in Hypertensive Patients With Type 2 Diabetes

A subgroup of patients with type 2 diabetes shows a clustering of abnormalities such as peripheral insulin resistance, hypertension, and microalbuminuria. To evaluate whether these traits reflect intrinsic disorders of cell function rather than in vivo environmental effects, we studied a group of 7 nondiabetic hypertensive subjects with an altered albumin excretion rate (AER) (HyMA+) and 3 groups of patients with type 2 diabetes: 7 with normal blood pressure and normal AER (DH−MA−), 7 with high blood pressure and normal AER (DH+MA−), and 7 with both high blood pressure and altered AER (DH+MA+). Glucose disposal was measured during an hyperinsulinemic clamp (40 mU · m2−1 · min−1) with primed deuterated [6.6 2H2] glucose infusion. In the same subjects, a skin biopsy was performed and the following parameters were investigated: glucose transport (as determined by [3H]2-deoxyglucose uptake); glycogen synthase activity (as determined by [14C] glucose incorporation from UDP-[U-14C] glucose into glycogen); glycogen phosphorylase activity (as measured by the incorporation of [U-14C]glucose 1-phosphate into glycogen); and total glycogen content. In vivo glucose disposal was significantly reduced in DH+MA− and DH+MA+, with respect to DH−MA−, HyMA+, and controls. Insulin-stimulated glucose transport was similar in the 3 groups of patients with diabetes. A significant reduction of intracellular glycogen content was observed in DH+MA− and DH+MA+ compared with DH−MA− in both basal and insulin-stimulated conditions, probably because of a major impairment of glycogen synthase activity. Glycogen phosphorylase activity did not show differences between the groups. These results suggest that (1) the combination of type 2 diabetes with hypertension and altered AER is associated with impaired insulin sensitivity, and (2) intrinsic, possibly genetic, factors may account for increased peripheral insulin resistance in hypertensive microalbuminuric patients with type 2 diabetes, pointing to the reduction of glycogen synthase activity as a shared common defect.