Renal Expression and Localization of the Receptor for (Pro)renin and Its Ligands in Rodent Models of Diabetes, Metabolic Syndrome, and Age-Dependent Focal and Segmental Glomerulosclerosis.

The (pro)renin receptor ((P)RR), a versatile protein found in various organs, including the kidney, is implicated in cardiometabolic conditions like diabetes, hypertension, and dyslipidemia, potentially contributing to organ damage. Importantly, changes in (pro)renin/(P)RR system localization during renal injury, a critical information base, remain unexplored. This study investigates the expression and topographic localization of the full length (FL)-(P)RR, its ligands (renin and prorenin), and its target cyclooxygenase-2 and found that they are upregulated in three distinct animal models of renal injury. The protein expression of these targets, initially confined to specific tubular renal cell types in control animals, increases in renal injury models, extending to glomerular cells. (P)RR gene expression correlates with protein changes in a genetic model of focal and segmental glomerulosclerosis. However, in diabetic and high-fat-fed mice, (P)RR mRNA levels contradict FL-(P)RR immunoreactivity. Research on diabetic mice kidneys and human podocytes exposed to diabetic glucose levels suggests that this inconsistency may result from disrupted intracellular (P)RR processing, likely due to increased Munc18-1 interacting protein 3. It follows that changes in FL-(P)RR cellular content mechanisms are specific to renal disease etiology, emphasizing the need for consideration in future studies exploring this receptor's involvement in renal damage of different origins.

Reduced Lipopolysaccharide-Binding Protein (LBP) Levels Are Associated with Non-Alcoholic Fatty Liver Disease (NAFLD) and Adipose Inflammation in Human Obesity.

Lipopolysaccharide (LPS) and its binding protein LBP have emerged as potential contributors to the progression from overweight/obesity to overt metabolic diseases and NAFLD. While LPS is known to activate hepatocyte inflammation, thus contributing toward NAFLD development, the role of LBP is more intricate, and recent data have shown that experimental reduction in hepatic LBP promotes NAFLD progression. In this cross-sectional investigation, we evaluated circulating LBP in relation to obesity, NAFLD, visceral adipose tissue (VAT) inflammation, and type 2 diabetes (T2D). We recruited 186 individuals (M/F: 81/105; age: 47 ± 10.4 years; BMI: 35.5 ± 8.6 kg/m2); a subgroup (n = 81) underwent bariatric surgery with intra-operative VAT and liver biopsies. LBP levels were higher in obese individuals than non-obese individuals but were inversely correlated with the parameters of glucose metabolism. Reduced LBP predicted T2D independent of age, sex, and BMI (p < 0.001). LBP levels decreased across more severe stages of hepatosteatosis and lobular inflammation, and were inversely associated with VAT inflammation signatures. In conclusion, LBP levels are increased in obese individuals and are associated with a more favorable metabolic profile and lower NAFLD/NASH prevalence. A possible explanation for these findings is that hepatic LBP production may be triggered by chronic caloric excess and facilitate LPS degradation in the liver, thus protecting these individuals from the metabolic consequences of obesity.

Neurotensin Gene rs2234762 C>G Variant Associates with Reduced Circulating Pro-NT Levels and Predicts Lower Insulin Resistance in Overweight/Obese Children.

Neurotensin (NT) is a small protein implicated in the regulation of energy balance which acts as both a neurotransmitter in the central nervous system and as a gastrointestinal peptide. In the gut, NT is secreted after fat ingestion and promotes the absorption of fatty acids. The circulating levels of its precursor, pro-NT, predicts the presence and development of metabolic and cardiovascular diseases. Despite the extensive knowledge on the dynamic changes that occur to pro-NT = after fat load, the determinants of fasting pro-NT are unknown. The aim of this study was to determine the possible genetic regulation of plasma pro-NT. The NT gene (NTS) was sequenced for potential functional variants, evaluating its entire genomic and potentially regulatory regions, in DNA from 28 individuals, stratified by low and high pro-NT levels. The identified variant differently distributed in the two pro-NT subgroups was genotyped in a cohort of nine hundred and thirty-two overweight/obese children and adolescents. A total of seven sequence variations across the NTS gene, none of them located in coding regions, were identified. The rs2234762 polymorphism, sited in the NTS gene promoter, was statistically more frequent in the lowest pro-NTS level group. Carriers of the rs2234762 variant showed lower pro-NT levels, after adjusting for sex, age, BMI, triglycerides and the Tanner stage. Having NTS rs2234762 predicted less pronounced insulin resistance at the 6.5-year follow-up with OR: 0.46 (0.216-0.983), at the logistic regression analysis adjusted for age, sex and BMI. In conclusion, the NTS rs2234762 gene variant is a determinant of reduced circulating pro-NT levels in overweight and obese children, which predisposes this group to a more favorable metabolic profile and a reduced insulin resistance later in life, independently from metabolic confounders.

Deep Resequencing of 9 Candidate Genes Identifies a Role for ARAP1 and IGF2BP2 in Modulating Insulin Secretion Adjusted for Insulin Resistance in Obese Southern Europeans.

Type 2 diabetes is characterized by impairment in insulin secretion, with an established genetic contribution. We aimed to evaluate common and low-frequency (1-5%) variants in nine genes strongly associated with insulin secretion by targeted sequencing in subjects selected from the extremes of insulin release measured by the disposition index. Collapsing data by gene and/or function, the association between disposition index and nonsense variants were significant, also after adjustment for confounding factors (OR = 0.25, 95% CI = 0.11-0.59, p = 0.001). Evaluating variants individually, three novel variants in ARAP1, IGF2BP2 and GCK, out of eight reaching significance singularly, remained associated after adjustment. Constructing a genetic risk model combining the effects of the three variants, only carriers of the ARAP1 and IGF2BP2 variants were significantly associated with a reduced probability to be in the lower, worst, extreme of insulin secretion (OR = 0.223, 95% CI = 0.105-0.473, p < 0.001). Observing a high number of normal glucose tolerance between carriers, a regression posthoc analysis was performed. Carriers of genetic risk model variants had higher probability to be normoglycemic, also after adjustment (OR = 2.411, 95% CI = 1.136-5.116, p = 0.022). Thus, in our southern European cohort, nonsense variants in all nine candidate genes showed association with better insulin secretion adjusted for insulin resistance, and we established the role of ARAP1 and IGF2BP2 in modulating insulin secretion.

Circulating pro-neurotensin levels predict bodyweight gain and metabolic alterations in children.

BACKGROUND AND AIMS: Neurotensin (NT) is an intestinal peptide released after fat ingestion, which regulates appetite and facilitates lipid absorption. Elevated plasma levels of its stable precursor pro-neurotensin (pro-NT) are associated with type 2 diabetes, obesity and cardiovascular mortality in adult populations; no data on pro-NT and metabolic disease are available in children. Aim of the study was to evaluate plasma pro-NT in relation to the presence of obesity in children, and to test if high pro-NT associates with the development of metabolic impairment later in life. METHODS AND RESULTS: For this longitudinal retrospective study, we studied 151 overweight/obese children undergoing metabolic evaluations at University of Cagliari, Italy. Pro-NT was also assessed in 46 normal-weight, age-, sex-comparable normal-weight children, selected as a reference group. At the baseline, pro-NT was comparable between overweight/obese and normal-weight children and correlated positively with age (p < 0.001), triglycerides (p < 0.001) and inversely with HDL levels (p = 0.008). Plasma pro-NT associated with high triglycerides with OR = 5.9 (95%CI: 1.24-28.1; p = 0.026) after adjustment for multiple confounders. At the 6.5-year follow-up, high basal pro-NT associated with impaired ß-cell function to compensate for insulin-resistance (disposition index: r = -0.19, p = 0.035) and predicted bodyweight increase, as indicated by percentage change of standard deviation score BMI (median(95%CI) = +20.8(+4.9-+27.5)% in the highest tertile), independently from age, sex, triglycerides and insulin-resistance (standardized ß = 0.24; p = 0.036). CONCLUSIONS: Elevated pro-NT levels in children are significantly associated with weight gain later in life and may represent a marker of susceptibility to metabolic impairment in presence of obesity.

Circulating dipeptidyl peptidase-4 is independently associated with the presence and severity of NAFLD/NASH in individuals with and without obesity and metabolic disease.

INTRODUCTION: Dipeptidyl peptidase 4 (DPP4) levels are associated to metabolic and cardiovascular diseases in humans; initial evidence reported a relationship between DPP4 and chronic liver diseases. Aim of this study was to investigate hepatic and systemic DPP4 levels/activity in relation to NAFLD/NASH in individuals with and without metabolic disease. METHODS: We recruited fifty-two obese individuals undergoing bariatric surgery and intra-operative liver biopsy at Sapienza University, Rome, Italy. The association between DPP4 levels/activity and NAFLD was also evaluated in 126 non-obese individuals recruited in the same setting. RESULTS: NAFLD patients had significantly higher circulating DPP4 activity than no-NAFLD in both the obese and non-obese cohorts; plasma DPP4 activity and levels linearly correlated with steatosis grade and inflammation at the liver biopsy. Hepatic DPP4 mRNA was not associated to either its circulating levels/activity or NAFLD. In the multivariate logistic regression analysis on all the study participants (n = 178), higher circulating DPP4 activity was associated with NAFLD independently of potential confounders with OR (95% CI): 3.5 (1.2-10.21), p = 0.022. CONCLUSIONS: This study demonstrates the coexistence of increased plasma DPP4 levels and activity in NAFLD. Circulating DPP4 measurement may represent a novel cost-effective strategy for NAFLD/NASH risk stratification and a potential tool for monitoring disease's progression in established NAFLD.

Association of Apelin Levels in Overweight-obese Children with Pubertal Development, but Not with Insulin Sensitivity: 6.5 Years Follow up Evaluation.

BACKGROUND: Obesity in youth is associated with increased risk of metabolic disorders. Adipose tissue hormones are involved in body-weight regulation. Among these, apelin is recognized as an insulin-sensitizer adipokine. Data on apelin levels in obese children and its relation to insulin-sensitivity are limited. OBJECTIVE: We aimed to evaluate apelin levels in relation to obesity and insulin sensitivity in a large cohort of overweight/obese children and adolescents. Furthermore, these youths were reevaluated after a median 6.5 years of follow-up, thus allowing assessing changes in apelin levels in relation to increasing age and weight changes. METHODS: Clinical data in 909 children and adolescents were collected between 2007 and 2010. Two hundred and one were reexamined at a median 6.5 years of follow-up. All subjects at baseline and at follow-up underwent an OGTT. Apelin levels were measured on sera by ELISA method. RESULTS: At baseline, lower apelin levels were associated with increasing age and puberty (Tanner ≥II 0.67 ± 0.96 ng/mL vs. Tanner I 0.89 ± 1.13 ng/mL, p < .002), but not with body-weight. At follow-up, apelin levels in the 201 subjects reexamined were significantly lower than at baseline (0.45 ± 0.77 ng/mL at follow-up, 0.68 ± 0.95 ng/mL baseline, p < .001), confirming the effects of age and puberty. Body-weight did not affect apelin levels. Multiple regression analysis confirmed that sex and puberty were associated with lower apelin levels, independently from age and insulin-sensitivity. CONCLUSIONS: Apelin levels decrease significantly with pubertal development, whilst body-weight in children and adolescents did not determine changes in apelin. Reduced levels of apelin in children and adolescents may therefore represent a necessary response to maintain the "physiological" insulin resistance of puberty. Abbreviations: ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; G: glucose; BMI: Body mass index; DBP: Diastolic blood pressure; ELISA: enzyme-linked immunosorbent assay; HDL-C: High-density lipoprotein-cholesterol; HOMA-B: Homeostatic model assessment for beta-cell function; HOMA-IR: Homeostatic model assessment of insulin-resistance; INS: Insulin; ISI: insulin-sensitivity index; LDL-C: Low-density lipoprotein cholesterol; NW: normal weight; OB: obese; OGTT: oral glucose tolerance test; OW: overweight; SBP: Systolic blood pressure; TC: Total cholesterol; TGs: Triglycerides.

Greater circulating DPP4 activity is associated with impaired flow-mediated dilatation in adults with type 2 diabetes mellitus.

BACKGROUND AND AIM: Dipeptidyl peptidase 4 (DPP4) is a key enzyme involved in the regulation of the incretin system exerted by cleaving the glucagon-like peptide 1 (GLP-1); the blockage of DPP4, exerted by the antidiabetic agents DPP4-inhibitors (DPP4-I), results in greater GLP-1 concentration and improved glycaemic control. DPP4 acts also as a pro-inflammatory molecule and mediates vascular damage in experimental models. The relationship between DPP4 activity and endothelial function in diabetes has not been explored yet. Aim of this study was to investigate systemic plasma DPP4 activity in relation to endothelial function in patients with type 2 diabetes mellitus (T2DM). METHODS AND RESULTS: Sixty-two T2DM individuals were recruited in our Diabetes outpatient clinics, Sapienza University, Rome, Italy. All participants underwent complete clinical work-up; endothelial function was evaluated by flow-mediated dilatation (FMD) test; plasma DPP4 activity was assessed by measuring the 7-amino-4-methylcoumarin (AMC) cleavage rate from the synthetic substrate H-glycyl-prolyl-AMC and compared with DPP4 activity measured in sixty-two age-, sex-, BMI-matched non-diabetic subjects. Patients with T2DM had significantly higher DPP4 activity than non-diabetic individuals (211,466 ± 87657 vs 158,087 ± 60267 nmol/min/ml, p < 0.001); in T2DM patients, greater DPP4 activity significantly correlated with lower FMD whereas was not associated with BMI and metabolic control. Greater systemic DPP4 activity was an independent predictor of reduced FMD after adjusting for age, gender and other confounders. CONCLUSIONS: Circulating DPP4 activity is increased in individuals with T2DM and associated with signs of endothelial dysfunction such as impaired FMD. DPP4 may negatively affect endothelial function through mechanisms beyond glucose homeostasis and metabolic control.

The Arg282Ser missense mutation in APOA5 gene determines a reduction of triglyceride and LDL-cholesterol in children, together with low serum levels of apolipoprotein A-V.

BACKGROUND: Apolipoprotein A-V (ApoA-V) is a recognized regulator of plasma triglycerides (TGs), and previous studies have shown associations between variants in APOA5 (apolipoprotein-A5) gene and high TG levels. Recently, a new association between the Arg282Ser missense mutation (rs778114184 G > T) in APOA5 gene and decreased triglyceride levels has been shown in an adult population from Sardinia. In this study we add further insight into the role of APOA5 by exploring whether this association begins early in life in children, or becomes manifest only in adulthood. We performed the genetic association analysis of APOA5 in a cohort of 925 overweight and obese children and adolescents from Sardinia, Italy, to see if the genetic burden is already at play before modifying risk factors are interacting. RESULTS: We identified 24 heterozygous subjects for the Arg282Ser variant and no homozygous subject. Here we show that the Arg282Ser mutation in APOA5 gene is associated with a significant reduction of TG (-15.5 mg/dl), total (-18.1 mg/dl) and LDL-cholesterol (-14.8 mg/dl) levels in overweight/obese children and adolescents, indicating that indeed this association appears early in life. Also, we observed a significant reduction in serum apoA-V levels in heterozygous children. CONCLUSIONS: Our data clearly show that the Arg282Ser mutation in APOA5 gene determines a reduction of TG, total and LDL-cholesterol and apolipoprotein A-V levels in overweight/obese children and adolescents, demonstrating that this mutation has the power to affect lipid levels already since childhood.

The vitamin D receptor functional variant rs2228570 (C>T) does not associate with type 2 diabetes mellitus.

AIM: Vitamin D acts through the binding to the vitamin D receptor (VDR). Several polymorphisms in VDR gene have been studied. Among these, the rs2228570 C>T (FokI) variant has been demonstrated to be functional, leading to a protein with a different size and activity. So far, genetic studies on the association between VDR gene rs2228570 single nucleotide polymorphism (SNP) and type 2 diabetes mellitus (T2DM) showed contradictory results. Thus, we performed an association study in a large cohort of adult Italian subjects with T2DM and in nondiabetic controls. MATERIALS AND METHODS: For this study, 1713 subjects, 883 T2DM patients and 830 controls, were genotyped for the polymorphism. All participants without a diagnosis of diabetes underwent oral glucose tolerance test (OGTT), with measurement of glucose and insulin levels. Indices of insulin resistance (Homeostatic model assessment of insulin resistance, insulin sensitivity index), secretion (homeostatic model assessment for beta-cell, corrected insulin response at 30 minutes) and disposition index were calculated. RESULTS: Genotype distributions and allele frequencies did not show difference between T2DM subjects and controls. We did not find significant differences among the three genotypes regarding gender, age, BMI, waist, hip, waist-to-hip ratio, and blood pressure. There were also no significant differences in lipid parameters, aspartate aminotransferase, and alanine aminotransferase levels. We tested for association with OGTT-derived data and surrogate indices of insulin resistance and secretion. We did not find significant differences among the genotypes in any of above-mentioned parameters. Furthermore, vitamin D levels were measured in a subgroup of subjects. We did not find significant differences among the genotypes. CONCLUSIONS: Our study does not provide evidence for the association of the rs2228570 polymorphism with T2DM in a Caucasian population.

The perilipin 2 (PLIN2) gene Ser251Pro missense mutation is associated with reduced insulin secretion and increased insulin sensitivity in Italian obese subjects.

BACKGROUND: Perilipin 2 (PLIN2), a member of the family of perilipin lipid droplets coating proteins, is very widely expressed. The Ser251Pro (rs35568725) missense mutation in exon 6 of PLIN2 gene was previously associated with increased lipid accumulation, decreased lipolysis and increased number of small lipid droplets per cell. Furthermore, the Pro251 mutation was associated with decreased plasma triglyceride and very low density lipoprotein concentrations in population studies. The aim of this study was to evaluate the effect of the Ser251Pro mutation of PLIN2 gene in a cohort with a higher predisposition to obesity-associated metabolic alterations, such as insulin resistance, decreased insulin-secretion, hyperglycaemia, and dyslipidaemia. METHODS: A large cohort (N = 1692) of Italian obese subjects (mean body mass index = 41 kg/m(2) ) was genotyped for the Ser251Pro mutation. All participants underwent oral glucose tolerance tests (OGTT), with measurement of glucose and insulin levels. Indices of insulin resistance and of insulin secretion were also calculated. Clinical and biochemical parameters were collected for all participants. RESULTS: We observed that insulin concentration was significantly reduced at 120 min after the administration of glucose in Pro251 allele carriers, whereas glucose levels were similar in Pro251 allele carriers and non-carriers throughout the OGTT. Furthermore, the CIR120 index of insulin secretion was significantly lower (P < 0.035) and the ISI index of insulin-sensitivity was significantly higher (P < 0.031) in carriers of the Pro251 allele. When we analysed men and women separately to test for gender-specific associations, we observed that in women insulin levels were significantly lower in Pro251 allele carriers compared with wild-type subjects throughout the whole OGTT. In men, we confirmed a significant reduction in insulin concentration only at 120 min after the OGTT. No significant differences between genotype groups regarding triglyceride levels and anyother clinical and metabolic parameters were observed. CONCLUSION: We observed a strong significant association between the PLIN2 Pro251 mutation and lower insulin secretion associated with an increased insulin sensitivity. Copyright © 2015 John Wiley & Sons, Ltd.

The COBLL1 C allele is associated with lower serum insulin levels and lower insulin resistance in overweight and obese children.

BACKGROUND: Childhood obesity is a growing epidemic worldwide, and it is associated with metabolic complications, such as insulin resistance. Recently, a genetic variation (rs7607980) in the COBLL1 gene has been associated with lower insulin resistance in adults. The aim of the study was to investigate if the association between COBLL1 rs7607980 genetic variant and lower insulin resistance was present early in life. METHODS: This sequence variant was genotyped in 878 overweight and obese children (mean age: 10 years) from Sardinia, Italy, from the outpatient clinic of the Pediatric Endocrine Unit, at the Regional Hospital for Microcitaemia in Cagliari. Insulin resistance was assessed by measurement of fasting circulating insulin levels before and after an oral glucose tolerance test and by HOMA-IR. RESULTS: The COBLL1 rs7607980 C allele was associated with lower fasting insulin and HOMA-IR levels (p = 0.002 and p = 0.035, respectively) in overweight and obese children. Importantly, lower insulin levels were also observed 2 h after oral glucose tolerance test in C allele carriers (p = 0.009). CONCLUSIONS: The present study shows for the first time, the association between COBLL1 rs7607980 C allele, lower serum insulin levels and lower insulin resistance in overweight and obese children.

Lack of effect of apolipoprotein C3 polymorphisms on indices of liver steatosis, lipid profile and insulin resistance in obese Southern Europeans.

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.

The 148M allele of the PNPLA3 gene is associated with indices of liver damage early in life.

BACKGROUND & AIMS: Childhood obesity is a growing problem worldwide. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity in children. Recently, the PNPLA3 gene I148M (rs738409) variant was demonstrated to be strongly associated with hepatic steatosis in obese adults. In this study we add further insight into the role of PNPLA3 by exploring whether this association begins early in life in obese children or becomes manifest only in adulthood. METHODS: Four hundred and seventy-five obese/overweight children and adolescents were genotyped for the I148M allele. Clinical and biochemical parameters were collected for all participants, including indices of hepatic injury, glucose tolerance and insulin resistance. Ultrasound imaging of the liver was obtained to assess the degree of steatosis in a subset of children. RESULTS: Carriers of two 148M alleles had a 52% increase in circulating ALT levels compared to carriers of two 148I alleles, with individuals with one 148M allele showing a 9.5% increase (p=0.001). AST concentration was also significantly higher in carriers of two and one M alleles (17.4% and 4%, respectively, p=0.022). A total of 36% of carries of two 148M alleles showed elevated ALT, defined as >30U/L, compared to only 10% of carriers of two 148I alleles (p<0.001). Liver steatosis was more prevalent in carriers of two 148M alleles. Glucose tolerance and insulin sensitivity were similar across all three genotypes. CONCLUSIONS: Our data show that the PNPLA3 gene I148M variant is associated with increased levels of ALT/AST in obese children and adolescents, suggesting that it confers genetic susceptibility to liver damage from a young age.

Granzyme B Expression in Visceral Adipose Tissue Associates With Local Inflammation and Glyco-Metabolic Alterations in Obesity.

Granzyme B (GrB) is a serine protease produced by immune and non-immune cells, able to promote multiple processes, like apoptosis, inflammation, extracellular matrix remodeling and fibrosis. GrB expression in visceral adipose tissue (VAT) was associated with tissue damage, local inflammation and insulin resistance in obesity murine model, but there is no data in humans. Aim of this study was to explore the expression of GrB in VAT from obese subjects in relation to adipose tissue injury, inflammation, metabolic alterations and GrB circulating levels. For this purpose, 85 obese individuals undergoing bariatric surgery and 35 healthy subjects (as control) were recruited at Sapienza University, Rome, Italy. Study participants underwent clinical work-up and routine biochemistry. mRNA expression of GrB in VAT and of a panel of VAT inflammatory markers was analyzed by real-time PCR. Serum GrB levels were measured by Elisa Affymetrix EBIO. We observed that 80% of obese patients expressed GrB mRNA in VAT, and GrB VAT expression was associated with the presence of local inflammation and glucose homeostasis alterations. Moreover, GrB serum levels, which were higher in obese subjects compared to non-obese healthy individuals, were associated with GrB expression in VAT and glyco-metabolic impairment. Our data show, for the first time in humans, that obese subjects with "sick" fat and altered glucose tolerance exhibit GrB expression in VAT, and suggest that GrB might contribute to obesity-related VAT inflammatory remodeling and glucose homeostasis dysregulation. Moreover, increased circulating GrB levels might represent a possible peripheral marker of VAT dysfunction in metabolic diseases.

Oral glucose tolerance test in Italian overweight/obese children and adolescents results in a very high prevalence of impaired fasting glycaemia, but not of diabetes.

BACKGROUND: Very few studies on glucose abnormalities in European overweight/obese children and adolescents are available, and scientific evidence on the value of standard oral glucose tolerance test (OGTT) in childhood is lacking. We therefore aimed to establish prevalence and features of impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and type 2 diabetes (T2D) in a large cohort of Italian overweight/obese children and adolescents and to assess the validity of standard OGTT in the paediatric population. METHODS: This is a 1-year observational study conducted on 736 (535 overweight/obese and 201 normal weight) consecutive paediatric patients attending the outpatient clinic of Paediatric Endocrine Unit. Clinical and biochemical parameters were collected for all participants. All overweight/obese subjects underwent OGTT. RESULTS: We observed a high prevalence of IFG (7.66%), more than twice that observed in other European children, but a low prevalence of IGT (3.18%) and T2D (0.18%). IFG was useless to predict IGT, having very low predictive value (7.3%) and sensitivity (17.6%). Compared to normal weight children, overweight/obese subjects showed significant differences in most metabolic and clinical parameters. In the overweight/obese group, having hyperglycaemia was associated to significantly higher blood pressure, homeostasis model assessment for insulin resistance, insulin and triglycerides. CONCLUSIONS: In our children, the prevalence of IFG is higher than that reported in other European cohorts, whereas T2D is rare. IFG appears not useful to detect IGT in childhood. Paediatric diagnostic cut-points, glucose load and timing of sampling need to be further validated to define glucose abnormalities in obese children that, compared with normal weight subjects, already are characterised by a different metabolic phenotype.

Identification of sequence variants in the UBL5 (ubiquitin-like 5 or BEACON) gene in obese children by PCR-SSCP: no evidence for association with obesity.

BACKGROUND: Childhood obesity has a strong genetic background. The human UBL5 (BEACON) gene has been suggested as a candidate gene for obesity. Previous studies in populations of different ethnicities have shown a significant association between UBL5 variants and measures of body fatness. AIMS: To identify mutations that may cause early-onset obesity we screened the UBL5 gene for sequence variations in a cohort of obese children who also had at least one obese parent (BMI >30 kg/m2) diagnosed before the age of 30 years. METHODS: We screened the UBL5 gene by PCR-SSCP and sequencing in a cohort (n=30) of obese children (mean age 6.9 +/- 3 yr), and then analysed SNPs by HRMA in a population of 160 obese and 140 lean individuals. RESULTS: Three sequence variations were detected: -422T>C in the 5'-UTR region, and -800T>A (rs10418248) and -860G>T in the promoter region. The SNPs -422 T>C in the 5'-UTR region and -860G>T have never been described before. These two SNPs did not co-segregate with obesity in relatives of the obese carriers. However, since in silico analysis of the -860G>T SNP region predicted a loss of the consensus binding site for RXR-alpha and RXR-beta, both involved in adipose cell regulation, we screened the -860G>T variant in a cohort of 300 individuals, 160 young obese (mean age 33 years) and 140 lean individuals. No differences in genotype distribution or in -860T allele frequencies were found between the two groups (1.8% vs. 1.4%, p = NS). In addition, no association was found between obesity and the previously described -800T>A SNP (rs10418248). CONCLUSION: Our data suggest that the UBL5 gene is unlikely to play a major role in the genetic susceptibility to early-onset obesity in our population.

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

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.

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

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 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.

Search for Genetic Variant in the Apelin Gene by Resequencing and Association Study in European Subjects.

AIMS: Apelin is a peptide produced and secreted by white adipose tissue. It is synthesized as preproapelin, a protein containing 77 aminoacids which is then cleaved to shorter active fragments. As an adipokine, apelin plays a role in the regulation of many biological functions, including body energy homeostasis and glucose metabolism, water balance, and immunity. We have recently demonstrated that subjects with type 2 diabetes (T2D) have significantly higher serum apelin levels compared with controls, and that these levels associate with fasting glucose, basal disposition index, age, and diagnosis of T2D. The first aim of this study was to search for sequence variants in the apelin gene (APLN), located on chromosome Xq25-q26.1 that may associate with serum levels of apelin. The second aim was to analyze the possible association between diabetes and diabetes-related traits and APLN variants. METHODS: We designed a two-step genetic association study. Step one consisted of an initial screen of 100 individuals selected from the extremes of the apelin distribution levels wherein we sequenced the APLN gene to identify common variants. In step two, the rs181301686 with a minor allele frequency >0.2 was genotyped in 917 individuals to explore its association with T2D and diabetes-related traits. RESULTS: Five sequence variations were found across the APLN gene. To test for association with apelin levels, the rs181301686 and rs2281069 single-nucleotide polymorphisms were genotyped in 256 subjects for whom serum apelin levels were available. No significant differences were observed in apelin levels between genotypes. Association analysis in 917 individuals did not show significant differences between APLN genotypes and diabetes and diabetes-related traits. CONCLUSIONS: Resequencing of the apelin gene in subjects stratified by low or high apelin levels identified five APLN variants in an European population. No association was found between the most frequent variant, diabetes, and metabolic parameters.