Stefano Romeo

Facilitation of muscle evoked responses after repetitive cortical stimulation in man

Abstract The technique of repetitive transcranial magnetic stimulation (rTMS) allows cortical motor areas to be activated by trains of magnetic stimuli at different frequencies and intensities. In this paper, we studied long-term neurophysiological effects of rTMS delivered to the motor cortex at 5 Hz with an intensity of 120% of motor threshold. Each stimulus of the train produced muscle-evoked potentials (MEPs) in hand and forearm muscles, which gradually increased in size from the first to the last shock. After the end of the train, the response to a single-test stimulus remained enhanced for 600–900 ms. In contrast, the train had no effect on the size of the MEPs evoked by transcranial electrical stimulation, while it suppressed H-reflexes in forearm muscles for 900 ms. We conclude that rTMS of these parameters increases the excitability of the motor cortex and that this effect outlasts the train for almost 1 s. At the spinal level, rTMS may increase presynaptic inhibition of Ia afferent fibers responsible for the H-reflex.

The adiponectin gene SNP+276G>T associates with early-onset coronary artery disease and with lower levels of adiponectin in younger coronary artery disease patients (age ≤50 years)

Adiponectin, an adipocyte-derived protein, is an essential modulator of insulin sensitivity and several studies suggest an important role of adiponectin in the processes leading to atherosclerosis, thus indicating the adiponectin gene as a potential candidate for coronary artery disease (CAD). In the present study we have studied the association between two single nucleotide polymorphisms (SNPs) (+45T>G and +276 G>T) of the adiponectin gene and CAD, looking also into the possible influence of these SNPs on adiponectin plasma levels. The SNPs were analysed in a first cohort of 595 subjects, 325 with CAD and 270 matched controls. We observed a significant association (p<0.001) between the SNP +276G>T in the adiponectin gene and CAD. In multivariate analysis, carriers of the +276G>T SNP had an odds ratio (OR) for CAD of 4.99 (p<0.0007). A strong interaction between the +276G>T SNP and age was also present (OR, 1.03; p<0.0001). The increase in CAD risk was most evident among individuals with early-onset CAD (age ≤50 years), whereas in older CAD subjects other factors, and not the adiponectin SNP, were the major determinants. Furthermore, in CAD subjects with early-onset disease this SNP was also a significant determinant of lower levels of serum adiponectin levels. This association resulted independent from the other variables known to be associated with CAD in our population, including sex, body mass index, high-density lipoprotein and Homeostasis Model Assessment for insulin resistance. To confirm the results the +276G>T SNP was analysed in a second cohort of CAD and controls. The difference between CAD and controls in the +276G>T SNP frequencies showed a similar trend as before, although not significant. The combination of the two cohorts (1,046 subjects: 580 CAD and 466 controls) showed a statistically significant association, particularly in CAD subjects with early-onset of disease. In addition, we confirmed that in younger CAD subjects the SNP was a significant determinant of lower levels of adiponectin. In view of these results, it could be speculated that the adiponectin gene variant, or a mutation in linkage with it, determines lower adiponectin gene expression, causing in turn an increased risk to develop insulin resistance, atherosclerosis and cardiovascular disease. The significant association of the adiponectin gene in subjects with early-onset CAD also suggests that that genetic factors for late-onset diseases may exert a greater influence in younger persons, when other risk factors are not as prevalent as in older age groups.

Effects of repetitive cortical stimulation on the silent period evoked by magnetic stimulation

Abstract The effects of repetitive transcranial stimulation (rTMS) on brain activity remain unknown. In healthy subjects, we studied the effects of rTMS on the duration of the cortical silent period (SP). Repetitive stimuli were delivered with a Cadwell High Speed Magnetic Stimulator and a figure-of-eight coil placed over the hand motor area. rTMS was delivered in trains of 11 or 20 stimuli at frequencies of 3 and 5 Hz and at stimulation intensities of 110 and 120% of motor threshold. The SP was recorded from the forearm muscles during a voluntary contraction (20% of maximum effort). rTMS delivered at a frequency of 3 and 5 Hz and intensities of 110 and 120% motor threshold prolonged the duration of the SP, without modifying either the size or the latency of the muscle-evoked potentials (MEP). A conditioning train of 11 stimuli at 3 Hz had no effect on the duration of the SP evoked by a single magnetic shock delivered 600 ms after the train. These findings show that rTMS increases the duration of the cortical SP, but does so only during the train of stimuli. rTMS probably changes the duration of the SP by facilitating cortical inhibitory interneurons.

The G972R variant of the insulin receptor substrate-1 (IRS-1) gene, body fat distribution and insulin-resistance.

Aims/hypothesis. Insulin resistance is recognised as the core factor in the pathogenesis of Type II (non-insulin-dependent) diabetes mellitus, hypertension and atherosclerosis. Several studies indicate the possible role of mutations of the insulin receptor substrate-1 (IRS-1) gene in the pathogenesis of insulin-resistance and suggest a possible interaction between the IRS-1 gene and obesity, either by an effect on the development of obesity or by causing or aggravating the obesity-associated insulin resistance. Therefore, the prevalence of the G972R mutation of the IRS-1 gene was compared in 157 non-diabetic obese subjects (BMI > 30 m/kg2) and in 157 lean subjects (BMI < 28 m/kg2). By investigating the relation between this IRS-1 mutation, measures of obesity and metabolic parameters, we explored the possible influence of this mutation on body fat distribution and insulin resistance. Methods. The G972R mutation was detected by PCR amplification and BstN-1 restriction enzyme digestion. Data were analysed by univariate and multivariate analysis. Results. The G972R allele was significantly more frequent in obese subjects than in lean subjects (p < 0.002); however, no difference was found between centrally and peripherally obese subjects. Obese G972R carriers had significantly higher BMI (p < 0.001), fasting insulin (p < 0.01), triglycerides (p < 0.03) and HOMAIR (p < 0.001) than obese non-carriers. No differences were observed between G972R carriers and non-carriers among control subjects. Multivariate analysis confirmed that the IRS-1 G972R mutation was significantly and independently associated with reduced insulin sensitivity (p < 0.009) in the obese group. Conclusion/interpretation. The G972R mutation of the IRS-1 gene associates with obesity, but not with fat distribution, in this Italian cohort, and within the obese subjects this IRS-1 variant strongly associates with metabolic parameters suggesting greater insulin-resistance. These findings indicate a possible interaction between the IRS-1 variant and obesity in worsening insulin sensitivity. [Diabetologia (2001) 44: 367–372]

The G-308A variant of the Tumor Necrosis Factor-α (TNF-α) gene is not associated with obesity, insulin resistance and body fat distribution

BackgroundTumor Necrosis Factor-α (TNF-α) has been implicated in the pathogenesis of insulin resistance and obesity. The increased expression of TNF-α in adipose tissue has been shown to induce insulin resistance, and a polymorphism at position -308 in the promoter region ofTNF-α has been shown to increase transcription of the gene in adipocytes. Aim of this study is to investigate the role of the G-308A TNFα variant in obesity and to study the possible influence of this mutation on body fat distribution and on measures of obesity (including Fat Free Mass, Fat Mass, basal metabolic rate), insulin resistance (measured as HOMAIR), and lipid abnormalities. The G-308A TNFα polymorphism has been studied in 115 patients with obesity (mean BMI 33.9 ± 0.5) and in 79 normal lean subjects (mean BMI 24.3 ± 0.3).MethodsThe G-308A variant, detected by PCR amplification and Nco-1 digestion, determines the loss of a restriction site resulting in a single band of 107 bp [the (A) allele].ResultsThe (A) allele frequencies of the G-308A TNFα polymorphism were 13.1% in the obese group and 14.6% in the lean subjects, with no significant difference between the two groups. Furthermore, no association was found with BMI classes, body fat distribution, HOMAIR, and metabolic abnormalities.ConclusionsOur study did not detect any significant association of the G-308A TNFα polymorphism with obesity or with its clinical and metabolic abnormalities in this population. Our data suggests that, in our population, the G-308A TNFα polymorphism is unlikely to play a major role in the pathogenesis of these conditions.

Cortical excitability in patients with essential tremor

We used transcranial magnetic stimulation in 10 patients with essential tremor and 8 matched healthy subjects. A round stimulating coil was placed over the vertex and electromyographic activity was recorded from the first dorsal interosseous muscle. Paired transcranial stimuli were delivered at interstimulus intervals of 3, 5, 20, 100, 150, and 200 ms. The intensity of the conditioning stimulus was 80% of motor threshold at short and 150% at long interstimulus intervals (ISIs). We also measured the silent period obtained after a single magnetic pulse delivered at 150% of motor threshold during a submaximal muscle contraction. Patients and controls had similar motor threshold and similar latencies. Paired magnetic stimuli given at short and long ISIs at rest, and during a voluntary muscle contraction, elicited similar responses in both groups. The silent period evoked by transcranial magnetic stimulation had a similar duration in patients with ET and controls. In conclusion, these findings suggest that patients with essential tremor have normal cortical motor area excitability.

The G972R variant of the insulin receptor substrate-1 (IRS-1) gene is associated with insulin resistance in "uncomplicated" obese subjects evaluated by hyperinsulinemic-euglycemic clamp.

Several association studies have indicated the insulin receptor substrate-1 (IRS-1) gene G972R variant as a genetic risk factor for insulin resistance, particularly in presence of obesity. A few studies have also suggested a possible effect of the G972R variant on insulin secretion. The aim of this study was to evaluate the role of the IRS-1 gene G972R variant in 61 subjects with “uncomplicated” obesity [i.e. without diabetes, hypertension, dyslipidemia, coronary artery disease (CAD)], studied by hyperinsulinemic-euglycemic clamp. The presence of the G972R variant, detected in real-time with LightCycler hybridisation probes, was related to the indexes of insulin sensitivity. Furthermore, the possible role of this variant on insulin secretion was studied by means of insulin release indexes derived from oral tolerance test (OGTT). Twenty-four point five percent (24.5%) (no.=15) of the obese subjects proved to be carriers of the G972R variant. M index (p<0.05), non-oxidative glucose (p<0.01), insulin clearance (p<0.03) and insulin sensitivity index (ISI) (p<0.005) were all significantly reduced in G972R carriers compared to non-carriers, indicating a significant reduction in insulin sensitivity in carriers of the variant. A logistic regression analysis confirmed the independent association between the G972R variant and reduced insulin sensitivity (p<0.03). The interaction between obesity and the G972R variant was also independently associated with a reduced insulin sensitivity (p<0.005), suggesting that obesity and G972R variant were more than additive in predicting insulin resistance. The analysis of insulin release indexes did not show any significant differences. Our results demonstrate the association of the G972R variant of the IRS-1 gene with reduced insulin sensitivity in obese subjects, and indicate a possible interaction between the IRS-1 variant and obesity in worsening of insulin sensitivity.

Search for genetic variants in the p66Shc longevity gene by PCR-single strand conformational polymorphism in patients with early-onset cardiovascular disease

BackgroundAmong the possible candidate genes for atherosclerosis experimental data point towards the longevity gene p66Shc. The p66Shc gene determines an increase of intracellular reactive oxygen species (ROS), affecting the rate of oxidative damage to nucleic acids. Knock-out p66Shc-/- mice show reduction of systemic oxidative stress, as well as of plasma LDL oxidation, and reduced atherogenic lesions. Thus, p66Shc may play a pivotal role in controlling oxidative stress and vascular dysfunction in vivo.MethodsWe searched for sequence variations in the p66Shc specific region of the Shc gene and its upstream promoter by PCR-SSCP in a selected group of early onset coronary artery disease (CAD) subjects (n. 78, mean age 48.5 ± 6 years) and in 93 long-living control subjects (mean age 89 ± 6 years).ResultsThe analysis revealed two variant bands. Sequencing of these variants showed two SNPs: -354T>C in the regulatory region of p66Shc locus and 92C>T in the p66 specific region (CH2). Both these variants have never been described before. The first substitution partially modifies the binding consensus sequence of the Sp1 transcription factor, and was detected only in two heterozygous carriers (1 CAD subjects and 1 control subject). The 92C>T substitution in the CH2 region consists in an amino acid substitution at codon 31 (proline to leucine, P31L), and was detected in heterozygous status only in one CAD subject. No subjects homozygous for the two newly described SNPs were found.ConclusionOnly two sequence variations in the p66Shc gene were observed in a total of 171 subjects, and only in heterozygotes. Our observations, in accordance to other studies, suggest that important variations in the p66Shc gene may be extremely rare and probably this gene is not involved in the genetic susceptibility to CAD.

Single-strand conformation polymorphism analysis of the glucose transporter gene GLUT1 in maturity-onset diabetes of the young

Abstract Maturity-onset diabetes of the young (MODY), an autosomal dominant, early-onset form of type-2 diabetes, is caused by mutations in five different genes all leading to defect(s) in the pancreatic β cell. However, some patients with this form of diabetes do not bear a mutation in any of the known (MODY1–MODY5) loci, a notion prompting the search for new MODY genes. Clinical and genetic data point toward a defect in β cell function in the majority of patients with MODY, and partners of the glucose-sensing device are reasonable functional candidates. The high-capacity glucose transporter GLUT2 has the ideal kinetic features for performing this task. However, complete GLUT2 deficiency in humans leads to hepato-renal glycogenosis (Fanconi-Bickel syndrome), and heterozygous GLUT2 mutations apparently behave in a recessive manner. Furthermore, in the human β cell GLUT1 mRNA is predominant when compared to GLUT2 and glucose influx appears to be largely mediated by this low-Km transporter. Thus, we looked for the presence of sequence variants by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) within the GLUT1 gene in 90 Italian pedigrees negative at the search for mutations in glucokinase (MODY2) and hepatocyte nuclear factor-1α (MODY3), the two genes responsible for about 60% of MODY cases in Italian children. We found three already described silent mutations and a new single base deletion in position -173 of the 5′ regulatory region. The -173delA variant, which was detected in the heterozygous or homozygous state in 30.8% of MODY patients examined and is located in a Nuclear Factor Y binding sequence, is not associated with hyperglycemia in affected relatives of MODY probands. In conclusion, it appears from these results that the glucose transporter gene GLUT1 is unlikely to play a major role in the etiology of MODY diabetes.

Ia presynaptic inhibition after muscle twitch in the arm

Contraction of upper limb muscles in healthy subjects was used to investigate presynaptic inhibition at spinal level. The H reflex recorded in the forearm flexor muscles in response to median nerve stimulation was depressed in amplitude from 400 ms to 1 s after a muscle twitch induced by transcranial stimulation, root stimulation, direct biceps stimulation, and triceps tendon tap. Stimulation of the cutaneous branch of musculocutaneous nerve, ipsilateral triceps and contralateral biceps, and biceps tendon tap did not alter H‐reflex size. Forearm flexor H‐reflex amplitude is therefore related to changes in proprioceptive inflow secondary to the biceps muscle twitch. Root and direct muscle stimulation both failed to reduce the size of the motor evoked potential (MEP) after transcranial magnetic stimulation, suggesting that the inhibition acts at presynaptic level. Attenuation of H‐reflex amplitude was related to the size of the muscle twitch and was less pronounced during an isometric twitch than during free joint movement. Our results suggest that the biceps muscle twitch produces long‐lasting inhibition of the Ia afferents from forearm flexor muscles. This is an important and a simple mechanism for suppressing proprioceptive input during movement.