Capillary contact points determine beta cell polarity, control secretion and are disrupted in the db/db mouse model of diabetes.

AIMS/HYPOTHESIS: Almost all beta cells contact one capillary and insulin granule fusion is targeted to this region. However, there are reports of beta cells contacting more than one capillary. We therefore set out to determine the proportion of beta cells with multiple contacts and the impact of this on cell structure and function. METHODS: We used pancreatic slices in mice and humans to better maintain cell and islet structure than in isolated islets. Cell structure was assayed using immunofluorescence and 3D confocal microscopy. Live-cell two-photon microscopy was used to map granule fusion events in response to glucose stimulation. RESULTS: We found that 36% and 22% of beta cells in islets from mice and humans, respectively, have separate contact with two capillaries. These contacts establish a distinct form of cell polarity with multiple basal regions. Both capillary contact points are enriched in presynaptic scaffold proteins, and both are a target for insulin granule fusion. Cells with two capillary contact points have a greater capillary contact area and secrete more, with analysis showing that, independent of the number of contact points, increased contact area is correlated with increased granule fusion. Using db/db mice as a model for type 2 diabetes, we observed changes in islet capillary organisation that significantly reduced total islet capillary surface area, and reduced area of capillary contact in single beta cells. CONCLUSIONS/INTERPRETATION: Beta cells that contact two capillaries are a significant subpopulation of beta cells within the islet. They have a distinct form of cell polarity and both contact points are specialised for secretion. The larger capillary contact area of cells with two contact points is correlated with increased secretion. In the db/db mouse, changes in capillary structure impact beta cell capillary contact, implying that this is a new factor contributing to disease progression.

Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ signalling to control glucose-dependent insulin secretion.

A developing understanding suggests that spatial compartmentalisation in pancreatic ß cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell subcellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact ß-cell structure, and enhances glucose-dependent Ca2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase (FAK), in regulating ß cells. Integrins and FAK are exclusively activated at the ß-cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose-dependent Ca2+ responses and insulin secretion. We conclude that FAK orchestrates the final steps of glucose-dependent insulin secretion within the restricted domain where ß-cell contact the islet capillaries.

Bioactivation of Encapsulation Membranes Reduces Fibrosis and Enhances Cell Survival.

Encapsulation devices are an emerging barrier technology designed to prevent the immunorejection of replacement cells in regenerative therapies for intractable diseases. However, traditional polymers used in current devices are poor substrates for cell attachment and induce fibrosis upon implantation, impacting long-term therapeutic cell viability. Bioactivation of polymer surfaces improves local host responses to materials, and here we make the first step toward demonstrating the utility of this approach to improve cell survival within encapsulation implants. Using therapeutic islet cells as an exemplar cell therapy, we show that internal surface coatings improve islet cell attachment and viability, while distinct external coatings modulate local foreign body responses. Using plasma surface functionalization (plasma immersion ion implantation (PIII)), we employ hollow fiber semiporous poly(ether sulfone) (PES) encapsulation membranes and coat the internal surfaces with the extracellular matrix protein fibronectin (FN) to enhance islet cell attachment. Separately, the external fiber surface is coated with the anti-inflammatory cytokine interleukin-4 (IL-4) to polarize local macrophages to an M2 (anti-inflammatory) phenotype, muting the fibrotic response. To demonstrate the power of our approach, bioluminescent murine islet cells were loaded into dual FN/IL-4-coated fibers and evaluated in a mouse back model for 14 days. Dual FN/IL-4 fibers showed striking reductions in immune cell accumulation and elevated levels of the M2 macrophage phenotype, consistent with the suppression of fibrotic encapsulation and enhanced angiogenesis. These changes led to markedly enhanced islet cell survival and importantly to functional integration of the implant with the host vasculature. Dual FN/IL-4 surface coatings drive multifaceted improvements in islet cell survival and function, with significant implications for improving clinical translation of therapeutic cell-containing macroencapsulation implants.

Identification of Novel Potential Type 2 Diabetes Genes Mediating ß-Cell Loss and Hyperglycemia Using Positional Cloning.

Type 2 diabetes (T2D) is a complex metabolic disease regulated by an interaction of genetic predisposition and environmental factors. To understand the genetic contribution in the development of diabetes, mice varying in their disease susceptibility were crossed with the obese and diabetes-prone New Zealand obese (NZO) mouse. Subsequent whole-genome sequence scans revealed one major quantitative trait loci (QTL), Nidd/DBA on chromosome 4, linked to elevated blood glucose and reduced plasma insulin and low levels of pancreatic insulin. Phenotypical characterization of congenic mice carrying 13.6 Mbp of the critical fragment of DBA mice displayed severe hyperglycemia and impaired glucose clearance at week 10, decreased glucose response in week 13, and loss of ß-cells and pancreatic insulin in week 16. To identify the responsible gene variant(s), further congenic mice were generated and phenotyped, which resulted in a fragment of 3.3 Mbp that was sufficient to induce hyperglycemia. By combining transcriptome analysis and haplotype mapping, the number of putative responsible variant(s) was narrowed from initial 284 to 18 genes, including gene models and non-coding RNAs. Consideration of haplotype blocks reduced the number of candidate genes to four (Kti12, Osbpl9, Ttc39a, and Calr4) as potential T2D candidates as they display a differential expression in pancreatic islets and/or sequence variation. In conclusion, the integration of comparative analysis of multiple inbred populations such as haplotype mapping, transcriptomics, and sequence data substantially improved the mapping resolution of the diabetes QTL Nidd/DBA. Future studies are necessary to understand the exact role of the different candidates in ß-cell function and their contribution in maintaining glycemic control.

Immunity-related GTPase induces lipophagy to prevent excess hepatic lipid accumulation.

BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association. METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2. RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage. CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets. LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.

Two Novel Candidate Genes for Insulin Secretion Identified by Comparative Genomics of Multiple Backcross Mouse Populations.

To identify novel disease genes for type 2 diabetes (T2D) we generated two backcross populations of obese and diabetes-susceptible New Zealand Obese (NZO/HI) mice with the two lean mouse strains 129P2/OlaHsd and C3HeB/FeJ. Subsequent whole-genome linkage scans revealed 30 novel quantitative trait loci (QTL) for T2D-associated traits. The strongest association with blood glucose [12 cM, logarithm of the odds (LOD) 13.3] and plasma insulin (17 cM, LOD 4.8) was detected on proximal chromosome 7 (designated Nbg7p, NZO blood glucose on proximal chromosome 7) exclusively in the NZOxC3H crossbreeding, suggesting that the causal gene is contributed by the C3H genome. Introgression of the critical C3H fragment into the genetic NZO background by generating recombinant congenic strains and metabolic phenotyping validated the phenotype. For the detection of candidate genes in the critical region (30-46 Mb), we used a combined approach of haplotype and gene expression analysis to search for C3H-specific gene variants in the pancreatic islets, which appeared to be the most likely target tissue for the QTL. Two genes, Atp4a and Pop4, fulfilled the criteria from our candidate gene approaches. The knockdown of both genes in MIN6 cells led to decreased glucose-stimulated insulin secretion, indicating a regulatory role of both genes in insulin secretion, thereby possibly contributing to the phenotype linked to Nbg7p In conclusion, our combined- and comparative-cross analysis approach has successfully led to the identification of two novel diabetes susceptibility candidate genes, and thus has been proven to be a valuable tool for the discovery of novel disease genes.

A collective diabetes cross in combination with a computational framework to dissect the genetics of human obesity and Type 2 diabetes.

To explore the genetic determinants of obesity and Type 2 diabetes (T2D), the German Center for Diabetes Research (DZD) conducted crossbreedings of the obese and diabetes-prone New Zealand Obese mouse strain with four different lean strains (B6, DBA, C3H, 129P2) that vary in their susceptibility to develop T2D. Genome-wide linkage analyses localized more than 290 quantitative trait loci (QTL) for obesity, 190 QTL for diabetes-related traits and 100 QTL for plasma metabolites in the outcross populations. A computational framework was developed that allowed to refine critical regions and to nominate a small number of candidate genes by integrating reciprocal haplotype mapping and transcriptome data. The efficiency of the complex procedure was demonstrated for one obesity QTL. The genomic interval of 35 Mb with 502 annotated candidate genes was narrowed down to six candidates. Accordingly, congenic mice retained the obesity phenotype owing to an interval that contains three of the six candidate genes. Among these the phospholipase PLA2G4A exhibited an elevated expression in adipose tissue of obese human subjects and is therefore a critical regulator of the obesity locus. Together, our broad and complex approach demonstrates that combined- and comparative-cross analysis exhibits improved mapping resolution and represents a valid tool for the identification of disease genes.

A computational biology approach of a genome-wide screen connected miRNAs to obesity and type 2 diabetes.

OBJECTIVE: Obesity and type 2 diabetes (T2D) arise from the interplay between genetic, epigenetic, and environmental factors. The aim of this study was to combine bioinformatics and functional studies to identify miRNAs that contribute to obesity and T2D. METHODS: A computational framework (miR-QTL-Scan) was applied by combining QTL, miRNA prediction, and transcriptomics in order to enhance the power for the discovery of miRNAs as regulative elements. Expression of several miRNAs was analyzed in human adipose tissue and blood cells and miR-31 was manipulated in a human fat cell line. RESULTS: In 17 partially overlapping QTL for obesity and T2D 170 miRNAs were identified. Four miRNAs (miR-15b, miR-30b, miR-31, miR-744) were recognized in gWAT (gonadal white adipose tissue) and six (miR-491, miR-455, miR-423-5p, miR-132-3p, miR-365-3p, miR-30b) in BAT (brown adipose tissue). To provide direct functional evidence for the achievement of the miR-QTL-Scan, miR-31 located in the obesity QTL Nob6 was experimentally analyzed. Its expression was higher in gWAT of obese and diabetic mice and humans than of lean controls. Accordingly, 10 potential target genes involved in insulin signaling and adipogenesis were suppressed. Manipulation of miR-31 in human SGBS adipocytes affected the expression of GLUT4, PPARγ, IRS1, and ACACA. In human peripheral blood mononuclear cells (PBMC) miR-15b levels were correlated to baseline blood glucose concentrations and might be an indicator for diabetes. CONCLUSION: Thus, miR-QTL-Scan allowed the identification of novel miRNAs relevant for obesity and T2D.

Diabetes prevalence in NZO females depends on estrogen action on liver fat content.

In humans and rodents, risk of metabolic syndrome is sexually dimorphic, with an increased incidence in males. Additionally, the protective role of female gonadal hormones is ostensible, as prevalence of type 2 diabetes mellitus (T2DM) increases after menopause. Here, we investigated the influence of estrogen (E2) on the onset of T2DM in female New Zealand obese (NZO) mice. Diabetes prevalence (defined as blood glucose levels >16.6 mmol/l) of NZO females on high-fat diet (60 kcal% fat) in week 22 was 43%. This was markedly dependent on liver fat content in week 10, as detected by computed tomography. Only mice with a liver fat content >9% in week 10 plus glucose levels >10 mmol/l in week 9 developed hyperglycemia by week 22. In addition, at 11 wk, diacylglycerols were elevated in livers of diabetes-prone mice compared with controls. Hepatic expression profiles obtained from diabetes-prone and -resistant mice at 11 wk revealed increased abundance of two transcripts in diabetes-prone mice: Mogat1, which catalyzes the synthesis of diacylglycerols from monoacylglycerol and fatty acyl-CoA, and the fatty acid transporter Cd36. E2 treatment of diabetes-prone mice for 10 wk prevented any further increase in liver fat content and reduced diacylglycerols and the abundance of Mogat1 and Cd36, leading to a reduction of diabetes prevalence and an improved glucose tolerance compared with untreated mice. Our data indicate that early elevation of hepatic Cd36 and Mogat1 associates with increased production and accumulation of triglycerides and diacylglycerols, presumably resulting in reduced hepatic insulin sensitivity and leading to later onset of T2DM.

Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice.

The adaptive response of skeletal muscle to exercise training is tightly controlled and therefore requires transcriptional regulation. DNA methylation is an epigenetic mechanism known to modulate gene expression, but its contribution to exercise-induced adaptations in skeletal muscle is not well studied. Here, we describe a genome-wide analysis of DNA methylation in muscle of trained mice (n = 3). Compared with sedentary controls, 2,762 genes exhibited differentially methylated CpGs (P < 0.05, meth diff >5%, coverage >10) in their putative promoter regions. Alignment with gene expression data (n = 6) revealed 200 genes with a negative correlation between methylation and expression changes in response to exercise training. The majority of these genes were related to muscle growth and differentiation, and a minor fraction involved in metabolic regulation. Among the candidates were genes that regulate the expression of myogenic regulatory factors (Plexin A2) as well as genes that participate in muscle hypertrophy (Igfbp4) and motor neuron innervation (Dok7). Interestingly, a transcription factor binding site enrichment study discovered significantly enriched occurrence of CpG methylation in the binding sites of the myogenic regulatory factors MyoD and myogenin. These findings suggest that DNA methylation is involved in the regulation of muscle adaptation to regular exercise training.

High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation.

Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP) MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS). HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.

Enhanced responsiveness to T-cell help causes loss of B-lymphocyte tolerance to a ß-cell neo-self-antigen in type 1 diabetes prone NOD mice.

Self-reactive B lymphocytes contribute to type 1 diabetes pathogenesis as APC and auto-Ab producers in NOD mice and humans. To shed light on the mechanisms responsible for the breakdown in B-lymphocyte self-tolerance to ß-cell Ag, we utilised a model whereby hen-egg lysozyme (HEL)-specific Ig Tg (IgHEL-Tg)-Tg B lymphocytes were allowed to develop in or were transferred into mice expressing the HEL Tg under an insulin promoter (insHEL-Tg). IgHEL-Tg B lymphocytes enhanced type 1 diabetes susceptibility of insHEL-Tg NOD mice. A comparison of the tolerogenic activity of IgHEL-Tg B lymphocytes with NOD and non-autoimmune-prone C57BL/6 genetic backgrounds showed that both were rendered anergic in the presence of insHEL when competing with polyclonal B lymphocytes. Nevertheless, NOD IgHEL-Tg B lymphocytes transferred into insHEL-Tg mice were more readily susceptible to rescue from anergy than their C57BL/6 counterparts, following provision of in vivo T-cell help. The different tolerogenic outcomes were an intrinsic property of B lymphocytes rather than being related to the quality of T-cell help, with the defective response being at least partially controlled by genes mapping to insulin-dependent diabetes (Idd) susceptibility loci on Chromosome 1 (Idd5) and 4 (Idd9/11).