Role of monogenic diabetes genes on beta cell function in Italian patients with newly diagnosed type 2 diabetes. The Verona Newly Diagnosed Type 2 Diabetes Study (VNDS) 13.

We tested the hypothesis that common genetic variability of beta-cell genes responsible for monogenic diabetes may affect beta cell function in type 2 diabetes mellitus (T2DM). We studied 794 drug- naïve GAD-negative patients with newly diagnosed T2DM (age: median=59 years; I.Q. range: 52-66; body mass index: 29.3 kg/m2; 26.6-32.9). Beta-cell function was assessed by state-of-art mathematical modeling of glucose/C-peptide curves during a 240'-300' frequently sampled oral glucose tolerance test, to provide the beta-cell responses to the rate of increase in glucose concentration (derivative control: DC) and to glucose concentration (proportional control: PC). Forty-two single nucleotide polymorphism (SNPs), selected to cover over 90% of common genetic variability, were genotyped in nine monogenic diabetes genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, KCNJ11 and ABCC8. Allelic variants of four SNPs (rs1303722 and rs882019 of GCK, rs7310409 of HNF1A and rs5219 of KCNJ11) were significantly associated with DC of beta-cell secretion (all P < 0.036). Allelic variants of four other SNPs (rs2868094 and rs6031544 of HNF4A, and rs1801262 and rs12053195 of NEUROD1) were associated with PC of beta-cell secretion (P < 0.02). In multivariate models, GCK, HNF1A and KCNJ11 SNPs explained 2.5% of the DC variability of beta-cell secretion, whereas HNF4A and NEUROD1 SNPs explained 3.6% of the PC variability of beta-cell secretion. We conclude that common variability of monogenic diabetes genes is significantly associated with an impaired beta-cell function in patients with newly diagnosed T2DM; thereby, these genes might be targeted by specific treatments in T2DM.

Haplotypes of the genes (GCK and G6PC2) underlying the glucose/glucose-6-phosphate cycle are associated with pancreatic beta cell glucose sensitivity in patients with newly diagnosed type 2 diabetes from the VNDS study (VNDS 11).

BACKGROUND: Elevated fasting plasma glucose has been associated with increased risk for development of type 2 diabetes (T2D). The balance between glucokinase (GCK) and glucose-6-phosphate catalytic subunit 2 (G6PC2) activity are involved in glucose homeostasis through glycolytic flux, and subsequent insulin secretion. AIM: In this study, we evaluated the association between the genetic variability of G6PC2 and GCK genes and T2D-related quantitative traits. METHODS: In 794 drug-naïve, GADA-negative, newly diagnosed T2D patients (VNDS; NTC01526720) we performed: genotyping of 6 independent tag-SNPs within GCK gene and 5 tag-SNPs within G6PC2 gene; euglycaemic insulin clamp to assess insulin sensitivity; OGTT to estimate beta-cell function (derivative and proportional control; DC, PC) by mathematical modeling. Genetic association analysis has been conducted using Plink software. RESULTS: Two SNPs within GCK gene (rs882019 and rs1303722) were associated to DC in opposite way (both p < 0.004). Two G6PC2 variants (rs13387347 and rs560887) were associated to both parameters of insulin secretion (DC and PC) and to fasting C-peptide levels (all p < 0.038). Moreover, subjects carrying the A allele of rs560887 showed higher values of 2h-plasma glucose (2hPG) (p = 0.033). Haplotype analysis revealed that GCK (AACAAA) haplotype was associated to decreased fasting C-peptide levels, whereas, the most frequent haplotype of G6PC2 (GGAAG) was associated with higher fasting C-peptide levels (p = 0.001), higher PC (ß = 6.87, p = 0.022) and the lower 2hPG (p = 0.012). CONCLUSION: Our findings confirmed the role of GCK and G6PC2 in regulating the pulsatility in insulin secretion thereby influencing insulin-signaling and leading to a gradual modulation in glucose levels in Italian patients with newly diagnosed T2D.

Association between MBOAT7 rs641738 polymorphism and non-alcoholic fatty liver in overweight or obese children.

BACKGROUND AND AIM: The association between non-alcoholic fatty liver (NAFL) and the variant rs641738 within the membrane bound O-acyltransferase domain-containing 7 (MBOAT7) gene is currently uncertain, especially in the paediatric population. We examined whether there is an association between this genetic variant and NAFL in a large multicentre, hospital-based cohort of Italian overweight/obese children. METHODS AND RESULTS: We studied 1760 overweight or obese children [mean age (SD): 11.1(2.9) years, z-body mass index (zBMI) 3.2(0.9)], who underwent ultrasonography for the diagnosis of NAFL. A subgroup of these children (n = 182) also underwent liver biopsy. Genotyping of the MBOAT7 rs641738 polymorphism was performed by TaqMan-Based RT-PCR system in each subject. Overall, 1131 (64.3%) children had ultrasound-detected NAFL; 528 (30%) had rs641738 CC genotype, 849 (48.2%) had rs641738 CT genotype, and 383 (21.8%) had rs641738 TT genotype, respectively. In the whole cohort, the interaction of MBOAT7 genotypes with zBMI was not associated with NAFL after adjustment for age, sex, serum triglycerides, serum alanine aminotransferase levels and patatin-like phospholipase domain-containing protein-3 (PNPLA3) genotype (adjusted-odds ratio 1.02 [95% CI 0.98-1.06]). Similarly, no association was found between MBOAT7 genotypes and NAFL after stratification by obesity status. MBOAT7 genotypes were not associated with the presence of non-alcoholic steatohepatitis or the stage of liver fibrosis in a subgroup of 182 children with biopsy-proven NAFLD. CONCLUSIONS: The results of this study did not show any significant contribution of MBOAT7 rs641738 polymorphism to the risk of having either NAFL on ultrasonography or NASH on histology in a large hospital-based cohort of Italian overweight/obese children.

Screening for non-alcoholic fatty liver disease using liver stiffness measurement and its association with chronic kidney disease and cardiovascular complications in patients with type 2 diabetes.

AIM: Despite the high prevalence and serious clinical implications of non-alcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM), NAFLD is usually overlooked during routine diabetes care. This study explored the proportion of NAFLD cases and increased liver fibrosis (LF), and the association between LF and either chronic kidney disease (CKD) or cardiovascular complications in T2DM patients. METHODS: The study included 137 patients with non-insulin-treated T2DM and no known liver disease consecutively attending our diabetes outpatients' service who underwent liver ultrasonography and liver stiffness measurement (LSM) using vibration-controlled transient elastography (FibroScan®). RESULTS: The proportion of patients with hepatic steatosis on ultrasonography was 73.7%, and the proportion with significant LF was 17.5% with an LSM cut-off ≥7kPa or 10.2% with an LSM cut-off ≥8.7kPa. The presence of CKD (estimated GFR <60mL/min/1.73m2 and/or abnormal albuminuria) increased significantly across LSM tertiles (from around 15% in tertile 1 to 45% in tertile 3). Cardiovascular complications (previous ischaemic heart disease, ischaemic stroke, permanent atrial fibrillation) also tended to increase across LSM tertiles (from around 15% to 30%). After adjusting for established risk factors and potential confounders, LSM tertile 3 remained significantly associated with an approximately threefold higher risk of prevalent CKD (adjusted OR: 3.28, 95% CI: 1.22-8.90; P=0.019), but not for cardiovascular complications. CONCLUSION: These results suggest that NAFLD and significant LF (as assessed by FibroScan®) are very commonly seen in T2DM outpatients with no known liver disease attending a secondary-care diabetes service, and that increased LF is associated with a greater proportion of chronic vascular complications, especially CKD.

Association between PNPLA3rs738409 polymorphism decreased kidney function in postmenopausal type 2 diabetic women with or without non-alcoholic fatty liver disease.

AIM: Evidence is emerging that PNPLA3 rs738409 polymorphism (the major genetic variant associated with susceptibility to non-alcoholic fatty liver disease [NAFLD]) is associated with chronic kidney disease (CKD) in non-diabetic individuals. Currently, little is known about this association in type 2 diabetic (T2DM) patients with and without NAFLD. METHODS: We studied 101 Caucasian post-menopausal women with T2DM, consecutively attending our diabetes outpatient service during a 3-month period. Glomerular filtration rate (eGFRCKD-EPI) was estimated using the CKD-Epidemiology Collaboration (CKD-EPI) equation, whilst albuminuria was measured with an immunonephelometric assay on morning spot urine samples. NAFLD was detected either by fatty liver index (FLI ≥ 60, n = 101) or by ultrasonography (n = 77). Genotyping was performed by TaqMan-Based RT-PCR system. RESULTS: Eight patients had G/G, 41 G/C and 52 C/C PNPLA3 rs738409 genotypes, and 21 (20.8%) patients had CKD (eGFRCKD-EPI < 60 mL/min/1.73 m2 or abnormal albuminuria). Compared to those with G/C or C/C genotypes, patients with G/G genotype had significantly lower eGFRCKD-EPI (63.7 ± 11 vs. 77.4 ± 17 vs. 81.9 ± 15 mL/min/1.73 m2, P = 0.014) and higher prevalence of CKD (50% vs. 24.4% vs. 13.5%, P = 0.04). After adjustment for age, duration of diabetes, haemoglobin A1c, HOMA-estimated insulin resistance, systolic blood pressure, hypertension treatment and FLI ≥ 60, rs738409 G/G genotype was independently associated with both lower eGFRCKD-EPI (ß coefficient: -15.5, 95% CI -26.0 to -5.0, P = 0.004) and higher risk of CKD (adjusted-odds ratio 8.05, 95% CI 1.26-41.4, P = 0.03). Similar results were found when we adjusted for hepatic steatosis on ultrasography (instead of FLI ≥ 60). CONCLUSION: Regardless of the presence of NAFLD and common cardio-renal risk factors, in post-menopausal women with T2DM, the G/G genotype of rs738409 in the PNPLA3 gene was strongly associated with lower eGFRCKD-EPI and higher prevalence of CKD.

Enantiomer discrimination illustrated by high-resolution crystal structures of the human nuclear receptor hRARgamma.

The human retinoic acid receptor (hRAR) is a member of the nuclear receptor superfamily that regulates the transcription of target genes in a ligand-dependent manner. The three hRAR isotypes are targets for retinoids that are used in the treatment of various diseases, including breast cancer and skin diseases. Drug efficiency and safety depend on the pharmacological activity of enantiomers, which can differ because of the chiral environment generated by the target. We report the crystal structures of the hRARgamma ligand-binding domain bound to two enantiomers, the active BMS270394 and the inactive BMS270395, solved at 1.6 A and 1.7 A resolution, respectively. The crystal structures reveal that in both enantiomers, the hydroxyl moiety attached to the chiral center forms a hydrogen bond to the Met-272 sulfur atom, thus imposing a conformation of BMS270395 that differs significantly from that observed for BMS270394 and other known retinoids. BMS270395 adopts an energetically unfavorable conformation, accounting for its inactivity; in contrast, the conformation of BMS270394 is close to an energy minimum. Our high-resolution data allow rationalization of enantiomer discrimination by the receptor and provide a model system for the pharmacological properties of enantiomeric pairs.

Two distinct actions of retinoid-receptor ligands.

Signalling by all-trans retinoic acid is mediated through RXR-RAR retinoid receptor heterodimers, in which RXR has been considered to act as a transcriptionally silent partner. However, we show here that in cultured NB4 (ref. 6) human acute promyelocytic leukaemia cells treated with either an RAR-alpha-selective agonist alone, or certain RAR-alpha antagonists in combination with an RXR agonist, receptor-DNA binding is induced in vivo, resulting in expression of the target genes of retinoic acid as well as acute promyelocytic leukaemia protein (PML) relocation to nuclear bodies and differentiation before apoptosis. These results indicate that RAR-alpha ligands can induce two separate events: one enables RXR-RAR-alpha heterodimers to bind to DNA in vivo and allows RXR agonists to act; the other induces transcriptional activity of RAR-alpha. The availability of receptor-specific synthetic retinoids that can induce distinct receptor functions has potential in extending the therapeutic repertoire of retinoids.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.