Farooq Syed

Microarray analysis of isolated human islet transcriptome in type 2 diabetes and the role of the ubiquitin–proteasome system in pancreatic beta cell dysfunction

To shed light on islet cell molecular phenotype in human type 2 diabetes (T2D), we studied the transcriptome of non-diabetic (ND) and T2D islets to then focus on the ubiquitin-proteasome system (UPS), the major protein degradation pathway. We assessed gene expression, amount of ubiquitinated proteins, proteasome activity, and the effects of proteasome inhibition and prolonged exposure to palmitate. Microarray analysis identified more than one thousand genes differently expressed in T2D islets, involved in many structures and functions, with consistent alterations of the UPS. Quantitative RT-PCR demonstrated downregulation of selected UPS genes in T2D islets and beta cell fractions, with greater ubiquitin accumulation and reduced proteasome activity. Chemically induced reduction of proteasome activity was associated with lower glucose-stimulated insulin secretion, which was partly reproduced by palmitate exposure. These results show the presence of many changes in islet transcriptome in T2D islets and underline the importance of the association between UPS alterations and beta cell dysfunction in human T2D.

β-Cell inflammation in human type 2 diabetes and the role of autophagy.

β‐Cell failure is crucial for the onset and progression of human type 2 diabetes, and a few studies have suggested that inflammation may play a role. Immune cell infiltration has been reported in subpopulations of islets in some cases of human type 2 diabetes, and altered gene expression of a few cytokines and chemokines has been observed in isolated islets and laser captured β‐cells from diabetic subjects. Recent observations on the links between inflammation, apoptosis and autophagy are putting the focus on the possibility that modulating the autophagic processes could protect the β‐cells from cytotoxicity induced by inflammatory mediators.

Mast cells infiltrate pancreatic islets in human type 1 diabetes

Aims/hypothesisBeta cell destruction in human type 1 diabetes occurs through the interplay of genetic and environmental factors, and is mediated by immune cell infiltration of pancreatic islets. In this study, we explored the role of mast cells as an additional agent in the pathogenesis of type 1 diabetes insulitis.MethodsPancreatic tissue from donors without diabetes and with type 1 and 2 diabetes was studied using different microscopy techniques to identify islet-infiltrating cells. The direct effects of histamine exposure on isolated human islets and INS-1E cells were assessed using cell-survival studies and molecular mechanisms.ResultsA larger number of mast cells were found to infiltrate pancreatic islets in samples from donors with type 1 diabetes, compared with those from donors without diabetes or with type 2 diabetes. Evidence of mast cell degranulation was observed, and the extent of the infiltration correlated with beta cell damage. Histamine, an amine that is found at high levels in mast cells, directly contributed to beta cell death in isolated human islets and INS-1E cells via a caspase-independent pathway.Conclusions/interpretationThese findings suggest that mast cells might be responsible, at least in part, for immune-mediated beta cell alterations in human type 1 diabetes. If this is the case, inhibition of mast cell activation and degranulation might act to protect beta cells in individuals with type 1 diabetes.

Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes

Aims/hypothesisPancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP).MethodsAffymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells.ResultsComparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes.Conclusions/interpretationThese studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Direct effects of rosuvastatin on pancreatic human beta cells.

The 3-hydroxy-methylglutaryl coenzyme A inhibitors statins are largely used for primary and secondary prevention of atherosclerotic cardiovascular disease in both non-diabetic and diabetic patients [1, 2]. However, recent work has suggested that statin therapy may be associated with increased risk of new onset diabetes and deterioration of glycemic control. The concern was initially raised in 2008, when increased incidence of diabetes among patients taking rosuvastatin in the JUPITER study was reported [3]. A successive meta-analysis of randomized placebo-controlled and standard care–controlled trials (19,140 subjects of whom 4,278 developed diabetes) demonstrated a 9 % increased risk of incident diabetes in statin-treated individuals [4]. The benefits of statin treatment largely exceed the diabetes hazard [5–7]; nevertheless, it is important to investigate the mechanisms through which these molecules might affect glucose homeostasis. In this study, we have assessed the direct action of rosuvastatin on isolated human islets. Insulin secretion, beta cell survival, ultrastructure and gene expression studies were performed.

The use of multilayer nano-encapsulation for the immunoprotection of isolated human islets: in vitro and in vivo studies.

1-OR Similar 3-Year-Mortality in Patients with STEMI and NSTEMI for Known as well as for Newly Diagnosed Diabetes—Results of the SWEETHEART Registry ANSELM K. GITT, PETER BRAMLAGE, STEFFEN SCHNEIDER, RALF ZAHN, DIETHELM TSCHÖPE, SWEETHEART-STUDY-GROUP, Ludwigshafen, Germany, Mahlow, Germany, Bad Oeynhausen, Germany Joint ESC/EASD guidelines recommend testing for diabetes (DM) using oral glucose tolerance test (OGTT) in patients with CAD. SWEETHEART enrolled 2,767 consecutive patients (pts) with STEMI or NSTEMI to identify newly diagnosed DM (new DM) and to document outcome. In pts without DM, OGTT was performed at day 4 after the MI. We examined the impact of known and new DM on 3-year-outcome. OGTT detected 16.0%.new DM in STEMI and 17.8% in NSTEMI. Pts with new DM were younger and suffered from less concomitant diseases. 3-year-mortality rates were high, both for known and for new DM, without differences between STEMI and NSTEMI. OGTT after acute MI identifi ed new DM in 16.0% of STEMI and 17.8% of NSTEMI-pts. For known and new DM, 3-year-mortality was similar for STEMI and NSTEMI.

From genotype to human β cell phenotype and beyond

Polygenic type 2 diabetes mellitus (T2DM) is a multi-factorial disease due to the interplay between genes and the environment. Over the years, several genes/loci have been associated with this type of diabetes, with the majority of them being related to β cell dysfunction. In this review, the available information on how polymorphisms in T2DM-associated genes/loci do directly affect the properties of human islet cells are presented and discussed, including some clinical implications and the role of epigenetic mechanisms.

Multi-layer nanoencapsulation of human pancreatic islets

the previously identifi ed T2D single-nucleotide polymorphisms (SNPs) and SNPs found in regions near identifi ed T2D SNPs were associated with T2D within the Singapore Chinese Health Study. Using an Affymetrix GW ASI SNP array, we successfully genotyped 507,509 SNPs in 4678 participants (2338 cases with physician-diagnosed T2D and 2340 controls with glycated hemoglobin < 6.0%). Imputation using 1000 genomes project data was conducted for a fi ne-mapping analysis. We selected 98 T2D-related SNPs (p<10-5) from the NHGRI GWAS Catalog for replication. Forty-four SNPs were genotyped and 54 SNPs were imputed with r2>0.80. Positive associations between counts of the reported risk allele and T2D status were found for 73 of the 98 T2D SNPs; of which 24 were signifi cant (p<.05). Combining the reported risk alleles of the 98 SNPs into an unweighted risk score we estimated a relative risk per allele of 1.025 (p=7.5×10-10). Individuals in the highest quintile of the risk allele distribution were at 1.6-fold greater risk (p=0.0001) of T2D compared with those in the lowest quintile. Conditional analysis was performed for SNPs within 50kb of an index GWAS SNP by fi tting both the nearby and the GWAS SNPs in logistic regression. Several potentially novel independent associations (remaining signifi cant after multiple correction) were indicated and may be refl ecting new signals in some of the same regions containing already known T2D SNPs. In conclusion, we replicated many of the previously reported diabetes-related SNPs and their joint effects in Singaporean Chinese and identifi ed potentially novel candidate SNPs that are associated with T2D risk in an ethnic group with a high risk of diabetes. Further replication of novel SNPs is planned.

Conformal coating by multilayer nano-encapsulation for the protection of human pancreatic islets: In-vitro and in-vivo studies

To improve the efficiency of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human pancreatic islets coated by multi-layer nano-encapsulation using differently charged polymers [chitosan and poly(sodium styrene sulfonate)] to obtain up to 9 layers. The islet coating (thickness: 104.2 ± 4.2 nm) was uniform, with ≥ 90% cell viability and well preserved beta- and alpha-cell ultrastructure. Nano-encapsulated islets maintained physiological glucose-stimulated insulin secretion by both static incubation and perifusion studies. Notably, palmitate- or cytokine-induced toxicity was significantly reduced in nano-coated islets. Xenotransplantation of nano-encapsulated islets under the kidney capsule of streptozotocin-induced C57Bl/6J diabetic mice allowed long term normal or near normal glycemia, associated with minimal infiltration of immune cell into the grafts, well preserved islet morphology and signs of re-vascularization. In summary, the multi-layer nano-encapsulation approach described in the present study provides a promising tool to effectively protect human islets both in-vitro andin-vivo conditions.