Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion.

OBJECTIVES: Genome-wide association studies (GWAS) have identified >100 loci independently contributing to type 2 diabetes (T2D) risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells. METHODS: The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-ßH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq) so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-ßH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function. RESULTS: We found that the candidate T2D susceptibility genes' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-ßH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19) with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6) with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-ßH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the expression of Prc1, Srr, Zfand6, and Zfand3 was found in mouse pancreatic islets with altered beta-cell function. CONCLUSIONS: This study showed the ability of post-GWAS functional studies to identify new genes and pathways involved in human pancreatic beta-cell function and in T2D pathophysiology.

Automated assessment of ß-cell area and density per islet and patient using TMEM27 and BACE2 immunofluorescence staining in human pancreatic ß-cells.

In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as ß-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and ß-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the ß-cells suggest that these proteins reflect the total number of functional insulin producing ß-cells. Additionally, ß-cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real ß-cell number per islet.

Genetic and functional assessment of the role of the rs13431652-A and rs573225-A alleles in the G6PC2 promoter that are strongly associated with elevated fasting glucose levels.

OBJECTIVE: Genome-wide association studies have identified a single nucleotide polymorphism (SNP), rs560887, located in a G6PC2 intron that is highly correlated with variations in fasting plasma glucose (FPG). G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit. This study examines the contribution of two G6PC2 promoter SNPs, rs13431652 and rs573225, to the association signal. RESEARCH DESIGN AND METHODS: We genotyped 9,532 normal FPG participants (FPG <6.1 mmol/l) for three G6PC2 SNPs, rs13431652 (distal promoter), rs573225 (proximal promoter), rs560887 (3rd intron). We used regression analyses adjusted for age, sex, and BMI to assess the association with FPG and haplotype analyses to assess comparative SNP contributions. Fusion gene and gel retardation analyses characterized the effect of rs13431652 and rs573225 on G6PC2 promoter activity and transcription factor binding. RESULTS: Genetic analyses provide evidence for a strong contribution of the promoter SNPs to FPG variability at the G6PC2 locus (rs13431652: ß = 0.075, P = 3.6 × 10(-35); rs573225 ß = 0.073 P = 3.6 × 10(-34)), in addition to rs560887 (ß = 0.071, P = 1.2 × 10(-31)). The rs13431652-A and rs573225-A alleles promote increased NF-Y and Foxa2 binding, respectively. The rs13431652-A allele is associated with increased FPG and elevated promoter activity, consistent with the function of G6PC2 in pancreatic islets. In contrast, the rs573225-A allele is associated with elevated FPG but reduced promoter activity. CONCLUSIONS: Genetic and in situ functional data support a potential role for rs13431652, but not rs573225, as a causative SNP linking G6PC2 to variations in FPG, though a causative role for rs573225 in vivo cannot be ruled out.

Efficacy and safety of basiliximab in kidney transplantation.

The efficacy and safety of basiliximab, in combination with different maintenance regimens, are extensively addressed in the available literature. Basiliximab reduces the incidence of acute rejection, allows a safe reduction of steroid dosage, and is associated with economic savings, although there is substantially no proof that basiliximab prolongs either patient or graft survival. Initial basiliximab administration entails a low-risk and is associated with fewer adverse events than T cell depleting agents. However, life-threatening reactions were reported following re-exposure to basiliximab in recipients who lost graft function early after transplantation and, therefore, discontinued all immunosuppressive agents.

Pancreas preservation with University of Wisconsin and Celsior solutions: a single-center, prospective, randomized pilot study.

BACKGROUND: Celsior is an extracellular-type, low-viscosity, preservation solution already used for heart, lung, liver, and kidney transplantation. We report the results of a single-center, prospective, randomized pilot study specifically designed to compare the safety profile of Celsior solution with University of Wisconsin (UW) solution in clinical pancreas transplantation. METHODS: A total of 105 consecutive procurements were randomized to graft preservation with UW (n=53) solution or Celsior (n=52) solution. The groups were comparable with regard to all donor and recipient characteristics. RESULTS: Five grafts were discarded and 100 grafts (50 UW vs. 50 Celsior) were transplanted. Mean cold and warm ischemia times were 11.0 +/- 2.1 hr and 37.2 +/- 6.0 min for UW compared with 10.8 +/- 1.8 hr and 38.1 +/- 5.9 min for Celsior (P =not significant). Delayed endocrine pancreas function was recorded in one graft preserved with UW solution. Eleven recipients (UW 12% vs. Celsior 10%, P =not significant) required a relaparotomy. The mean serum levels of glucose, amylase, and lipase remained comparable between the study arms at equivalent intervals after transplantation. One recipient died with functioning grafts in each study arm; two further grafts were lost to arterial thrombosis (Celsior) and chronic rejection (UW), respectively. Actuarial 1-year patient and graft survival rates overlapped in the two study arms (98% and 96%, respectively). CONCLUSIONS: Within the range of cold ischemia time reported in this study, UW and Celsior solutions have similar safety profiles for pancreas preservation.

A benefit-risk assessment of basiliximab in renal transplantation.

Interleukin-2 (IL-2) and its receptor (IL-2R) play a central role in T lymphocyte activation and immune response after transplantation. Research on the biology of IL-2R allowed the identification of key signal transduction pathways involved in the generation of proliferative and antiapoptotic signals in T cells. The alpha-chain of the IL-2R is a specific peptide against which monoclonal antibodies have been raised, with the aim of blunting the immune response by means of inhibiting proliferation and inducing apoptosis in primed lymphocytes. Indeed, basiliximab, one of such antibodies, has proved to be effective in reducing the episodes of acute rejection after kidney and pancreas transplantation. The use of basiliximab was associated with a significant reduction in the incidence of any treated rejection episodes after kidney transplantation in the two major randomised studies (placebo 52.2% vs basiliximab 34.2% at 6 months, European study; placebo 54.9% vs basiliximab 37.6% at 1 year, US trial). Basiliximab and equine antithymocyte globulin (ATG) administration resulted in a similar rate of biopsy-proven acute rejection at 6 months (19% for both) and at 12 months (19% and 20%, respectively). The use of basiliximab appears not to be associated with an increased incidence of adverse events as compared with placebo in immunosuppressive regimens, including calcineurin inhibitors, mycophenolate mofetil or azathioprine and corticosteroids, and its safety profile is superior to ATG. Moreover, a similar occurrence of infections is noted in selected studies (65.5% after basiliximab vs 65.7% of controls), including cytomegalovirus infection (17.3% vs 14.5%), and cytokine-release syndrome is not observed. Finally, economic analysis demonstrated lower costs of overall treatment in patients treated with basiliximab. Therefore, the use of basiliximab entails a very low risk, allows safe reduction of corticosteroid dosage and reduces the short- and mid-term rejection rates. However, the improvement in the long-term survival of kidney grafts in patients treated according to modern immunosuppressive protocols is still to be demonstrated. These conclusions are based on a systematic review of the scientific literature, indexed on Medline database, concerning the mechanism of action, therapeutic activity, safety and pharmacoeconomic evaluation of basiliximab in renal transplantation.