Background

Apelin is an adipokine that plays a role in the regulation of glucose homeostasis and in obesity. The relationship between apelin serum concentration and dysmetabolic conditions such as type 2 diabetes (T2D) is still controversial. Aims of our study are: 1) determine the circulating levels of apelin in a large cohort of Italian subjects with T2D, T1D and in non-diabetic controls; 2) identify putative metabolic determinants of modified apelin concentrations, in order to search possible mechanism of apelin control; 3) investigate changes in apelin levels in response to sharp modifications of glucose/insulin metabolism in T2D obese subjects before and 3 days after bariatric surgery.

Methods

We recruited 369 subjects, 119 with T2D, 113 with T1D and 137 non-diabetic controls. All subjects underwent a complete clinical examination, including anthropometric and laboratory measurements. Serum apelin levels were determined by EIA (immunoenzyme assay).

Results

Patients with T2D had significantly higher serum apelin levels compared to controls (1.23±1.1 ng/mL vs 0.91±0.7 ng/mL, P<0.001) and to T1D subjects (0.73±0.39 ng/mL, P<0.001). Controls and T1D subjects did not differ significantly in apelin levels. Apelin concentrations were directly associated with fasting blood glucose (FBG), body mass index (BMI), basal Disposition Index (DI-0), age, and diagnosis of T2D at bivariate correlation analysis. Multiple regression analysis confirmed that diagnosis of T2D, basal DI-0 and FBG were all determinants of serum apelin levels independently from age and BMI. Bariatric surgery performed in a subgroup of obese diabetic subjects (n = 12) resulted in a significant reduction of apelin concentrations compared to baseline levels (P = 0.01).

Conclusions

Our study demonstrates that T2D, but not T1D, is associated with increased serum apelin levels compared to non-diabetic subjects. This association is dependent on impaired glucose homeostasis, and disappears after bariatric surgery, providing further evidence regarding the relationship between apelin and the regulation of glucose metabolism.

Background

Hepatic steatosis in HCV patients has been postulated as a risk factor associated with a higher frequency of fibrosis and cirrhosis. A single genetic variant, PNPLA3 I148M, has been widely associated with increased hepatic steatosis. Previous studies of the PNPLA3 I148M sequence variant in HCV infected individuals have reported an association between this variant and prevalence of steatosis, fibrosis, and cirrhosis. To evaluate the impact of PNPLA3 I148M variant on metabolic traits and treatment response in HCV genotype 2 and 3 infected patients.

Methods

Three hundred and eighty-two treatment naïve HCV genotype 2 or 3 infected patients were included in a phase III, open label, randomized, multicenter, investigator-initiated trial (the NORDynamIC study), in which pretreatment liver biopsies were mandatory. PNPLA3I148M genotyping was performed in a total of 359 Caucasian patients.

Results

In HCV genotype 2 infected patients carrying the PNPLA3 148M allele, there was significantly increased insulin resistance (P = 0.023) and lower viral load (P = 0.005) at baseline as well as the first seven days of antiviral treatment. These results were not observed in HCV genotype 3 infected patients.

Conclusions

Our results suggest a possible association between the PNPLA3 148M allele and insulin resistance as well as baseline viral load in HCV genotype 2, but not in genotype 3.

Background

Obesity is highly associated with elevated serum triglycerides, hepatic steatosis and type 2 diabetes (T2D). The I148M (rs738409) genetic variant of patatin-like phospholipase domain-containing 3 gene (PNPLA3) is known to modulate hepatic triglyceride accumulation, leading to steatosis. No association between PNPLA3 I148M genotype and T2D in Europeans has been reported. Aim of this study is to examine the relationship between PNPLA3 I148M genotypes and serum triglycerides, insulin resistance and T2D susceptibility by testing a gene-environment interaction model with severe obesity.

Methods and Findings

PNPLA3 I148M was genotyped in a large obese cohort, the SOS study (n = 3,473) and in the Go-DARTS (n = 15,448), a T2D case-control study. Metabolic parameters were examined across the PNPLA3 I148M genotypes in participants of the SOS study at baseline and at 2- and 10-year follow up after bariatric surgery or conventional therapy. The associations with metabolic parameters were validated in the Go-DARTS study. Serum triglycerides were found to be lower in the PNPLA3 148M carriers from the SOS study at baseline and from the Go-DARTS T2D cohort. An increased risk for T2D conferred by the 148M allele was found in the SOS study (O.R. 1.09, 95% C.I. 1.01-1.39, P = 0.040) and in severely obese individuals in the Go-DARTS study (O.R. 1.37, 95% C.I. 1.13-1.66, P = 0.001). The 148M allele was no longer associated with insulin resistance or T2D after bariatric surgery in the SOS study and no association with the 148M allele was observed in the less obese (BMI<35) individuals in the Go-DARTS study (P for interaction  = 0.002). This provides evidence for the obesity interaction with I48M allele and T2D risk in a large-scale cross-sectional and a prospective interventional study.

Conclusions

Severely obese individuals carrying the PNPLA3 148M allele have lower serum triglyceride levels, are more insulin resistant and more susceptible to T2D. This study supports the hypothesis that obesity-driven hepatic lipid accumulation may contribute to T2D susceptibility.

Obesity is recognized as a major health problem worldwide. Genetic factors play a major role in obesity, and genomewide association studies have provided evidence that several common variants within the fat mass- and obesity-associated (FTO) gene are significantly associated with obesity. Very limited data is available on FTO in the Italian population. Aims of our study are to investigate: (1) the association of FTO gene SNPs rs9939609 and rs9930506 with body mass index (BMI) and obesity-related parameters in a large cohort (n = 752) of Italian obese subjects; (2) the association between the two FTO SNPs and age of onset of obesity. Our results demonstrate a strong association between FTO SNPs rs9939609 (P < 0.043) and rs9930506 (P < 0.029) with BMI in the Italian population. FTO rs9930506 was significantly associated with higher BMI in a G allele dose-dependent manner (BMI + 1.4 kg/m2 per G allele). We also observed that the association with BMI of the two FTO variants varied with age, with the carriers of the risk alleles developing an increase in body weight earlier in life. In conclusion, our study further demonstrates a role of the genetic variability in FTO on BMI in a large Italian population.

Background

Apolipoprotein C3 (APOC3) is a component of triglyceride-rich lipoproteins, and APOC3 rs2854116 and rs2854117 polymorphisms have been associated with non-alcoholic fatty liver disease, hypertriglyceridaemia, and insulin-resistance.

Objective

To determine if the APOC3 variants alter the susceptibility of obese subjects to develop liver damage, hypertrigliceridaemia, and insulin-resistance.

Methods

The study was carried out on 585 unrelated obese Italians (median body mass index BMI = 41 kg/m2) who were genotyped for the rs2854116 and rs2854117 variants. All participants underwent oral glucose tolerance tests (OGTT), with measurement of glucose, insulin, lipid parameters. Indices of insulin-resistance (HOMA and ISI) were calculated. Alanine transaminase (ALT) and aspartate transaminase (AST) were used as markers of liver injury.

Results

The study subjects were divided into two groups: those homozygous for the wild-type alleles at both SNPs (-482C and -455T alleles) and those who were carriers of at least one variant allele or both (-482T, -455C or both). Also each SNP was analysed independently. No significant differences were found in ALT and AST levels and in the lipid profile between the two groups. Insulin concentrations, glucose tolerance and insulin sensitivity were similar in the two groups.

Conclusion

We did not identify any significant association between APOC3 polymorphisms and fatty liver disease, lipids, and insulin-resistance in obese subjects, thus not confirming the suggested role of these APOC3 gene sequence variants.

Resequencing genes provides the opportunity to assess the full spectrum of variants that influence complex traits. Here we report the first application of resequencing to a large population (n = 3,551) to examine the role of the adipokine ANGPTL4 in lipid metabolism. Nonsynonymous variants in ANGPTL4 were more prevalent in individuals with triglyceride levels in the lowest quartile than in individuals with levels in the highest quartile (P = 0.016). One variant (E40K), present in ~3% of European Americans, was associated with significantly lower plasma levels of triglyceride and higher levels of high-density lipoprotein cholesterol in European Americans from the Atherosclerosis Risk in Communities Study and in Danes from the Copenhagen City Heart Study. The ratio of nonsynonymous to synonymous variants was higher in European Americans than in African Americans (4:1 versus 1.3:1), suggesting population-specific relaxation of purifying selection. Thus, resequencing of ANGPTL4 in a multiethnic population allowed analysis of the phenotypic effects of both rare and common variants while taking advantage of genetic variation arising from ethnic differences in population history.

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem of unknown etiology that varies in prevalence among ethnic groups. To identify genetic variants contributing to differences in hepatic fat content, we performed a genome-wide association scan of nonsynonymous sequence variations (n=9,229) in a multiethnic population. An allele in PNPLA3 (rs738409; I148M) was strongly associated with increased hepatic fat levels (P=5.9×10−10) and with hepatic inflammation (P=3.7×10−4). The allele was most common in Hispanics, the group most susceptible to NAFLD; hepatic fat content was > 2-fold higher in PNPLA3-148M homozygotes than in noncarriers. Resequencing revealed another allele associated with lower hepatic fat content in African-Americans, the group at lowest risk of NAFLD. Thus, variation in PNPLA3 contributes to ethnic and inter-individual differences in hepatic fat content and susceptibility to NAFLD.

Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates the disposition of circulating triglycerides (TG) by inhibiting lipoprotein lipase (LPL). Here we examine the steps involved in the synthesis and post-translational processing of ANGPTL4, and the effects of a naturally occurring sequence variant (E40K) that is associated with lower plasma TG levels in humans. Expression of the wild-type and mutant proteins in HEK-293A cells indicated that ANGPTL4 formed dimers and tetramers in cells prior to secretion and cleavage of the protein. After cleavage at a canonical proprotein convertase cleavage site (161RRKR164), the oligomeric structure of the N-terminal domain was retained whereas the C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of cleavage did not interfere with oligomerization of ANGPTL4 or with its ability to inhibit LPL, whereas mutations that prevented oligomerization severely compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the E40K substitution was synthesized and processed normally, but no monomers or oligomers of the N-terminal fragments accumulated in the medium; medium from these cells failed to inhibit LPL activity. Parallel experiments performed in mice recapitulated these results. Our findings indicate that oligomerization, but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K substitution destabilizes the protein after secretion, preventing the extracellular accumulation of oligomers and abolishing the ability of the protein to inhibit LPL activity.

Background

Characterization of disease-associated balanced translocations has led to the discovery of genes responsible for many disorders, including syndromes that include various forms of diabetes mellitus. We studied a man with unexplained maturity onset diabetes of the young (MODY)-like diabetes and an apparently balanced translocation [46,XY,t(7;10)(q22;p12)] and sought to identify a novel diabetes locus by characterizing the translocation breakpoints.

Results

Mutations in coding exons and splice sites of known MODY genes were first ruled out by PCR amplification and DNA sequencing. Fluorescent in situ hybridization (FISH) studies demonstrated that the translocation did not disrupt two known diabetes-related genes on 10p12. The translocation breakpoints were further mapped to high resolution using FISH and somatic cell hybrids and the junctions PCR-amplified and sequenced. The translocation did not disrupt any annotated transcription unit. However, the chromosome 10 breakpoint was 220 kilobases 5' to the Membrane Protein, Palmitoylated 7 (MPP7) gene, which encodes a protein required for proper cell polarity. This biological function is shared by HNF4A, a known MODY gene. Databases show MPP7 is highly expressed in mouse pancreas and is expressed in human islets. The translocation did not appear to alter lymphoblastoid expression of MPP7 or other genes near the breakpoints.

Conclusion

The balanced translocation and MODY-like diabetes in the proband could be coincidental. Alternatively, the translocation may cause islet cell dysfunction by altering MPP7 expression in a subtle or tissue-specific fashion. The potential roles of MPP7 mutations in diabetes and perturbed islet cell polarity in insulin secretion warrant further study.

The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoprotein-derived fatty acids between sites of fat storage (adipose tissue) and oxidation (heart and skeletal muscle). Here we used a reverse genetic strategy to test the hypothesis that 4 angiopoietin-like proteins (ANGPTL3, -4, -5, and -6) play key roles in triglyceride (TG) metabolism in humans. We re-sequenced the coding regions of the genes encoding these proteins and identified multiple rare nonsynonymous (NS) sequence variations that were associated with low plasma TG levels but not with other metabolic phenotypes. Functional studies revealed that all mutant alleles of ANGPTL3 and ANGPTL4 that were associated with low plasma TG levels interfered either with the synthesis or secretion of the protein or with the ability of the ANGPTL protein to inhibit LPL. A total of 1% of the Dallas Heart Study population and 4% of those participants with a plasma TG in the lowest quartile had a rare loss-of-function mutation in ANGPTL3, ANGPTL4, or ANGPTL5. Thus, ANGPTL3, ANGPTL4, and ANGPTL5, but not ANGPTL6, play nonredundant roles in TG metabolism, and multiple alleles at these loci cumulatively contribute to variability in plasma TG levels in humans.

Background

Among 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.

Methods

We 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).

Results

The 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.

Conclusion

Only 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.

Background

Current evidence demonstrates that positive family history and several alterations in lipid metabolism are all important risk factors for coronary artery disease (CAD). All lipid abnormalities themselves have genetic determinants. Thus, objective of this study was to determine whether 6 genetic variants potentially related to altered lipid metabolism were associated with CAD and with lipid abnormalities in an Italian population. These genetic variables were: apolipoprotein E (Apo E), Apo AI, Apo CIII, Apo B, lipoprotein lipase (LPL) and the hepatic lipase (LIPC) genes. Furthermore, an 8 years prospective analysis of clinical cardiovascular events was related to the various genetic markers.

Methods

102 subjects with established coronary artery disease and 104 unrelated normal subjects were studied. CAD Patients were followed up for 8 years, and clinical CAD outcomes (a second coronary angioplasty (PTCA), myocardial infarction, coronary artery by-pass graft (CABG), cardiovascular deaths), available from 60 subjects, were related to the genetic variants by multiple regression analysis. Results. Of the six lipid loci studied (for a total of 11 polymorphisms) only the apolipoprotein E, Apo B and LIPC polymorphisms distinguished between case and controls. However, multivariate analysis accounting for clinical and metabolic predictors of CAD showed that only the ApoB Xba1 and ApoE4 polymorphism associated with CAD in this Italian population. When lipid parameters were related to genotypes, the ApoE, ApoB, and LIPC gene polymorphisms were associated to various markers of dyslipidaemia in the CAD patients, confirming previous reports. When the occurrence of a second cardiovascular event was related to genotypes, an independent role was observed for the LIPC gene T202T variant.

Conclusions

variation in LIPC (hepatic lipase) gene associates with clinical outcomes in Italian patients with established CAD. Further studies on the LIPC gene in CAD patients are warranted, in particular looking at the possible influences on clinical outcomes.

Background

Tumor 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).

Methods

The 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].

Results

The (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.

Conclusions

Our 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.