Grazia Fini

Serum Resistin, Cardiovascular Disease and All-Cause Mortality in Patients with Type 2 Diabetes

Background High serum resistin has been associated with increased risk of cardiovascular disease in the general population, Only sparse and conflicting results, limited to Asian individuals, have been reported, so far, in type 2 diabetes. We studied the role of serum resistin on coronary artery disease, major cardiovascular events and all-cause mortality in type 2 diabetes. Methods We tested the association of circulating resistin concentrations with coronary artery disease, major cardiovascular events (cardiovascular death, non-fatal myocardial infarction and non-fatal stroke) and all-cause mortality in 2,313 diabetic patients of European ancestry from two cross-sectional and two prospective studies. In addition, the expression of resistin gene (RETN) was measured in blood cells of 68 diabetic patients and correlated with their serum resistin levels. Results In a model comprising age, sex, smoking habits, BMI, HbA1c, and insulin, antihypertensive and antidyslipidemic therapies, serum resistin was associated with coronary artery disease in both cross-sectional studies: OR (95%CI) per SD increment = 1.35 (1.10–1.64) and 1.99 (1.55–2.55). Additionally, serum resistin predicted incident major cardiovascular events (HR per SD increment = 1.31; 1.10–1.56) and all-cause mortality (HR per SD increment = 1.16; 1.06–1.26). Adjusting also for fibrinogen levels affected the association with coronary artery disease and incident cardiovascular events, but not that with all cause-mortality. Finally, serum resistin was positively correlated with RETN mRNA expression (rho = 0.343). Conclusions This is the first study showing that high serum resistin (a likely consequence, at least partly, of increased RETN expression) is a risk factor for cardiovascular disease and all-cause mortality in diabetic patients of European ancestry.

Evidence for genetic epistasis in human insulin resistance: the combined effect of PC-1 (K121Q) and PPARγ2 (P12A) polymorphisms

Insulin resistance is believed to be under the control of several genes often interacting each other. However, whether genetic epistasis does in fact modulate human insulin sensitivity is unknown. In 338 healthy unrelated subjects from Sicily, all nondiabetic and not morbidly obese, we investigated whether two gene polymorphisms previously associated with insulin resistance (namely PC-1 K121Q and PPARγ2 P12A) affect insulin sensitivity by interacting. PC-1 X121Q subjects showed higher level of fasting glucose, lower insulin sensitivity (by both the Matsuda insulin sensitivity index and M values at clamp, the latter performed in a subgroup of 113 subjects representative of the overall cohort) and higher insulin levels during the oral glucose tolerance test (OGTT) than PC-1 K121K subjects. In contrast, no difference in any of the measured variables was observed between PPARγ2 P12P and X12A individuals. The deleterious effect of the PC-1 X121Q genotype on each of these three variables was significant and entirely dependent upon the coexistence of the PPARγ2 P12P genotype. Among PPARγ2 P12P carriers also fasting insulin and glucose levels during OGTT were higher in PC-1 X121Q than in K121K individuals. In contrast, no deleterious effect of the PC-1 X121Q genotype was observed among PPARγ2 X12A carriers; rather, in these subjects a lower body mass index and consequently lower fasting insulin level was observed in PC-1 X121Q than in K121K carriers. Overall, a significant interaction between the two genes was observed on body mass index, insulin levels (both fasting and after OGTT) and both insulin sensitivity (i.e., insulin sensitivity index and M value) and insulin secretion (i.e., HOMA-B%) indexes.

Circulating high molecular weight adiponectin isoform is heritable and shares a common genetic background with insulin resistance in nondiabetic White Caucasians from Italy: evidence from a family-based study.

Abstract.  Menzaghi C, Salvemini L, Paroni G, De Bonis C, Mangiacotti D, Fini G, Doria A, Di Paola R, Trischitta V (IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy, Joslin Diabetes Center and Harvard Medical School, Boston, MA, USA, “Sapienza” University; IRCCS Casa Sollievo della Sofferenza‐Mendel Institute, Rome, Italy). Circulating HMW adiponectin isoform is heritable and shares a common genetic background with insulin resistance in nondiabetic White Caucasians from Italy: evidence from a family‐based study. J Intern Med 2010; 267: 287–294.

Circulating adiponectin and cardiovascular mortality in patients with type 2 diabetes mellitus: evidence of sexual dimorphism.

BackgroundThe pathogenesis of cardiovascular (CV) mortality, whose rate is increased in type 2 diabetes, is poorly understood.While high serum adiponectin is associated with increased CV mortality in the general population, no data are available in type 2 diabetes.We here investigated whether this counterintuitive association was observable also in diabetic patients and whether it was sex-specific.MethodsThree prospective cohorts were analyzed: 1) Gargano Heart Study (GHS; 359 patients, 58 events/1,934 person-years; py); 2) Health Professional Follow-up Study (HPFS; 833 men, 146 events/10,024 py); 3) Nurses’ Health Study (NHS; 902 women, 144 events/15,074 py).ResultsIn GHS serum adiponectin predicted CV mortality in men (hazard ratio, HR, and 95% CI per standard deviation, SD, increment = 1.54, 1.19-2.01), but not women (HR = 0.98, 0.48-2.01).Circulating adiponectin predicted CV mortality in men from HPFS (HR = 1.44, 1.21-1.72), but not in women from NHS (HR = 1.08, 0.86-1.35), used as replication samples. In a pooled analysis, HRs were 1.47 (1.27-1.70) in 1,075 men and 1.07 (0.86-1.33) in 1,019 women (p for HRs heterogeneity across sexes = 0.018).ConclusionsThis is the first report showing that high circulating adiponectin predicts increased CV mortality in men, but not in women with type 2 diabetes. Further studies are necessary to unravel the mechanisms through which adiponectin influences CV mortality in a sex-specific manner.

ENPP1 Q121 Variant, Increased Pulse Pressure and Reduced Insulin Signaling, and Nitric Oxide Synthase Activity in Endothelial Cells

Objective—Insulin resistance induces increased pulse pressure (PP), endothelial dysfunction (ED), and reduced bioavailability of endothelium-derived nitric oxide (NO). The genetic background of these 3 cardiovascular risk factors might be partly common. The ENPP1 K121Q polymorphism is associated with insulin resistance and cardiovascular risk. Methods and Results—We investigated whether the K121Q polymorphism is associated with increased PP in white Caucasians and with ED in vitro. In 985 individuals, (390 unrelated and 595 from 248 families), the K121Q polymorphism was associated with PP (P=8.0×10−4). In the families, the Q121 variant accounted for 0.08 of PP heritability (P=9.4×10−4). This association was formally replicated in a second sample of 475 individuals (P=2.6×10−2) but not in 2 smaller samples of 289 and 236 individuals (P=0.49 and 0.21, respectively). In the individual patients’ data meta-analysis, comprising 1985 individuals, PP was associated with the Q121 variant (P=1.2×10−3). Human endothelial cells carrying the KQ genotype showed, as compared to KK cells, reduced insulin-mediated insulin receptor autophosphorylation (P=0.03), Ser473-Akt phosphorylation (P=0.03), and NO synthase activity (P=0.003). Conclusions—Our data suggest that the ENPP1 Q121 variant is associated with increased PP in vivo and reduced insulin signaling and ED in vitro, thus indicating a possible pathogenic mechanism for the increased cardiovascular risk observed in ENPP1 Q121 carriers.

GALNT2 Expression Is Reduced in Patients with Type 2 Diabetes: Possible Role of Hyperglycemia

Impaired insulin action plays a major role in the pathogenesis of type 2 diabetes, a chronic metabolic disorder which imposes a tremendous burden to morbidity and mortality worldwide. Unraveling the molecular mechanisms underlying insulin resistance would improve setting up preventive and treatment strategies of type 2 diabetes. Down-regulation of GALNT2, an UDPN-acetyl-alpha-D-galactosamine polypeptideN-acetylgalactosaminyltransferase-2 (ppGalNAc-T2), causes impaired insulin signaling and action in cultured human liver cells. In addition, GALNT2 mRNA levels are down-regulated in liver of spontaneously insulin resistant, diabetic Goto-Kakizaki rats. To investigate the role of GALNT2 in human hyperglycemia, we measured GALNT2 mRNA expression levels in peripheral whole blood cells of 84 non-obese and 46 obese non-diabetic individuals as well as of 98 obese patients with type 2 diabetes. We also measured GALNT2 mRNA expression in human U937 cells cultured under different glucose concentrations. In vivo studies indicated that GALNT2 mRNA levels were significantly reduced from non obese control to obese non diabetic and to obese diabetic individuals (p<0.001). In vitro studies showed that GALNT2 mRNA levels was reduced in U937 cells exposed to high glucose concentrations (i.e. 25 mmol/l glucose) as compared to cells exposed to low glucose concentration (i.e. 5.5 mmol/l glucose +19.5 mmol/l mannitol). In conclusion, our data indicate that GALNT2 is down-regulated in patients with type 2 diabetes and suggest that this association is, at least partly, secondary to hyperglycemia. Further studies are needed to understand whether GALNT2 down-regulation plays a pathogenic role in maintaining and/or aggravating the metabolic abnormalities of diabetic milieu.

Serum Resistin and Kidney Function: A Family-Based Study in Non-Diabetic, Untreated Individuals

Background High serum resistin levels have been associated with kidney dysfunction. Most of these studies have been carried out in individuals with severe kidney impairment, diabetes, cardiovascular disease and related treatments. Thus, the observed association might have been influenced by these confounders. Our aim was to study the relationship between serum resistin, urinary albumin/creatinine ratio (ACR) and glomerular filtration rate (GFR) in a family-based sample, the Gargano Family Study (GFS) of 635 non diabetic, untreated Whites. Methods A linear mixed effects model and bivariate analyses were used to evaluate the phenotypic and genetic relations between serum resistin and both ACR and eGFR. All analyses were adjusted for sex, age, age squared, BMI, systolic blood pressure, smoking habits and physical exercise. Results After adjustments, resistin levels were slightly positively associated with ACR (β±SE = 0.049±0.023, p = 0.035) and inversely related to eGFR (β±SE = −1.43±0.61, p = 0.018) levels. These associations remained significant when either eGFR or ACR were, reciprocally, added as covariates. A genetic correlation (ρg = −0.31±0.12; adjusted p = 0.013) was observed between resistin and eGFR (but not ACR) levels. Conclusion Serum resistin levels are independently associated with ACR and eGFR in untreated non-diabetic individuals. Serum resistin and eGFR share also some common genetic background. Our data strongly suggest that resistin plays a role in modulating kidney function.

Relationship between ADIPOQ gene, circulating high molecular weight adiponectin and albuminuria in individuals with normal kidney function: evidence from a family-based study

Aims/hypothesisInsulin resistance is associated with reduced serum adiponectin and increased albuminuria levels. Thus, one would anticipate an inverse relationship between circulating adiponectin and albuminuria. However, several studies have described a ‘paradoxical’ elevation of serum adiponectin in patients with elevated albuminuria. These findings may have been confounded by the presence of diseases and related treatments known to affect circulating adiponectin and albuminuria. We therefore studied the relationship between circulating adiponectin and albuminuria in the absence of such confounders.MethodsTo this purpose, the relationship between adiponectin isoforms and albumin:creatinine ratio (ACR) was investigated in a family-based sample of 634 non-diabetic untreated white individuals with normal kidney function. We also investigated whether the two variables share a common genetic background and addressed the specific role of the gene encoding adiponectin on that background by genotyping several ADIPOQ single nucleotide polymorphisms (SNPs).ResultsACR was directly associated with high molecular weight (HMW) adiponectin isoform (p = 0.024). The two variables shared some genetic correlation (ρg = 0.38, p = 0.04). ADIPOQ promoter SNP rs17300539 was associated with HMW adiponectin (p = 4.8 × 10−5) and ACR (p = 0.0027). The genetic correlation between HMW adiponectin and ACR was no longer significant when SNP rs17300539 was added to the model, thus reinforcing the role of this SNP in determining both traits.Conclusions/interpretationOur study shows a positive, independent correlation between HWM adiponectin and ACR. ADIPOQ variability is associated with HMW adiponectin and ACR, and explains some of the common genetic background shared by these traits, thus suggesting that ADIPOQ and HMW adiponectin modulate albuminuria levels.

Molecular Dissection of the VHL Gene in Solitary Capillary Hemangioblastoma of the Central Nervous System

Capillary hemangioblastomas (HGBs) of the CNS occur either sporadically or as part of the von Hippel-Lindau (VHL) syndrome. Molecular characterizations of the VHL gene in sporadic HGBs at the somatic level have been limited to date. We investigated the VHL gene in 57 patients most of whom (55 [96%] of 57) had a solitary CNS HGB at the time of surgery. Tissues from 23 HGBs of these patients (2 VHL related and 21 unrelated) were also investigated at genetic and epigenetic levels. Two of the 51 patients with apparently sporadic HGBs and no additional evidence of VHL (∼4%) were found to have a germline VHL gene mutation; both of these patients subsequently developed evidence of VHL syndrome. Somatic VHL gene mutations were found in 11 (52%) of the 21 non-VHL-related cases. A germline mutation was identified in 5 (84%) of 6 VHL-associated HGBs; double gene inactivation was observed in tumor tissue from VHL syndrome patients. Seven different previously unreported VHL gene alterations (6 somatic and 1 germline) were identified; double hits were identified in 7 (12%) of 57 cases. Our findings confirm the usefulness of VHL gene analysis at the germline level in patients who present with apparently solitary HGB. Moreover, the genetic and epigenetic VHL gene investigations performed support a key role for functional alterations of the VHL gene in sporadic neuraxial HGB.

Lack of evidence for interaction between APM1 and PPARgamma2 genes in modulating insulin sensitivity in nondiabetic Caucasians from Italy.

Dear Sir, The genetic background of insulin resistance is believed to be polygenic with several genes simultaneously involved and each other interacting. We recently described the association between two APM1 SNPs (i.e. +45T>G and +276G>T, considered either singly or in combination as TG haplotype) and insulin resistance-related features in nondiabetic Caucasians from centre-east coast Italy [1]. The +276G>T SNP also predicts serum adiponectin concentrations in the same population [2] and coronary artery disease in type 2 diabetes patients from the same region [3]. The functional missense Pro12Ala SNP in the PPARc2 gene has been proposed to have a broad impact on the risk of insulin resistance and type 2 diabetes [4]. It has been recently described that the APM1 +45T>G SNP interacts with the PPARc2 Pro12Ala SNP in modulating insulin resistance in subjects from a Japanese and Chinese hypertensive family cohort [5]. The possibility of such an interaction is indeed reinforced by the presence of a functional PPAR responsive element in the human APM1 promoter [6] and by the observation that troglitazone, an insulin sensitizer, which bind and activate PPARc2, increases adiponectin serum concentrations and ameliorates insulin resistance in type 2 diabetes patients [7]. The aim of the present work was to verify whether the +45T>G and/or +276G>T SNPs, interact with the PPARc2 Pro12Ala variant in modulating insulin resistance and related features also in Caucasians. The study group included 574 unrelated, nondiabetic subjects recruited among the employees of the Scientific Institute Casa Sollievo della Sofferenza (San Giovanni Rotondo, Italy) who had fasting plasma glucose <7 mmol L and were not taking medications known to interfere with insulin sensitivity and/or glucose metabolism. The study was approved by the local Ethical Committee. Anthropometric measures, mean blood pressure and fasting blood glucose, serum insulin and adiponectin were measured as previously described [1, 2]. APM1 +45T>G and +276G>T and PPARc2 Pro12Ala polymorphisms were detected as indicated [1, 2]. The effect of genotypes on metabolic traits and other continuous variables were assessed by linear regression analysis using a model including age and gender. The effect of modification between APM1 TG haplotype and PPARc2 Pro12 Ala was tested by introducing an interaction term in the regression model. Fasting insulin, HOMAIR and serum adiponectin were analysed in the logarithms. A P value of <0.05 was considered as significant. SPSS for Windows v. 10.1. (SPSS Inc., Chicago, IL, USA) was used. SNPs +45T>G and +276G>T, considered in combination as the TG haplotype were significantly associated with blood pressure, serum insulin and adiponectin levels and the insulin resistance index HOMAIR (Table 1) as previously described in a 30% smaller sample [1, 2]. At variance, Pro12Ala polymorphism had no effect per se on insulin resistance and related variables (Table 1). The same trend of differences observed in the whole population between subjects being or not homozygotes for the APM1 TG haplotype on blood pressure, serum insulin and HOMAIR levels were maintained in PPARc2 Pro12Pro carriers (n 1⁄4 485) and PPARc2 X12Ala (n 1⁄4 65) individuals as well (Table 2), thus clearly indicating no interaction between APM1 and PPARc2 genes in modulating these variables. Differences in serum adiponectin levels tended to show an opposite trend in the two PPARc2 subgroups; however, also in this case, no evidence for a significant gene–gene interaction was obtained (Table 2). Worth nothing, with the present sample size, we have 80% power to detect a significant difference (P 1⁄4 0.05) of 31 pmol L in serum insulin and of 1.1 in HOMAIR index values. Therefore, although the real pathophysiological significance of smaller Journal of Internal Medicine 2005; 257: 315–317