Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ∼80 fF that plateaued within ∼200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K d values were 6 and 29 μM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1−/− mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1−/− mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl− into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.

A plethora of candidate genes have been identified for complex polygenic disorders, but the underlying disease mechanisms remain largely unknown. We explored the pathophysiology of type 2 diabetes (T2D) by analyzing global gene expression in human pancreatic islets. A group of coexpressed genes (module), enriched for interleukin-1-related genes, was associated with T2D and reduced insulin secretion. One of the module genes that was highly overexpressed in islets from T2D patients is SFRP4, which encodes secreted frizzled-related protein 4. SFRP4 expression correlated with inflammatory markers, and its release from islets was stimulated by interleukin-1β. Elevated systemic SFRP4 caused reduced glucose tolerance through decreased islet expression of Ca(2+) channels and suppressed insulin exocytosis. SFRP4 thus provides a link between islet inflammation and impaired insulin secretion. Moreover, the protein was increased in serum from T2D patients several years before the diagnosis, suggesting that SFRP4 could be a potential biomarker for islet dysfunction in T2D.

SUMMARY There are multiple mechanisms of maintaining tolerance in the gut that protect the intestine from excessive inflammatory response to intestinal microorganisms. We report here that all four mammalian Peptidoglycan Recognition Proteins (PGRPs or Pglyrps) protect the host from colitis induced by dextran sulfate sodium (DSS). Pglyrp1−/−, Pglyrp2−/−, Pglyrp3−/−, and Pglyrp4−/− mice are all more sensitive than wild type (WT) mice to DSS-induced colitis due to changes to more inflammatory gut microflora, higher production of interferon-γ and interferon-inducible genes, and increase in NK cells in the colon upon initial exposure to DSS, which leads to severe hyperplasia of the lamina propria, loss of epithelial cells, and ulceration in the colon. Thus in WT mice PGRPs protect the colon from early inflammatory response and loss of the barrier function of intestinal epithelium by promoting normal bacterial flora and by preventing damaging production of interferon-γ by NK cells in response to injury.

Concerted activation of different voltage-gated Ca 2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca V 2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca V 2.3-knockout (Ca V 2.3 -/-) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca 2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca V 2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca 2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca V 2.3 -/-β cells. Ca V 2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca V 2.3 -/-mouse. We propose a specific role for Ca V 2.3 Ca 2+ channels in second-phase insulin release, that of mediating the Ca 2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation. IntroductionSystemic glucose tolerance is orchestrated by the regulated release of insulin and glucagon from the β and α cells of the pancreatic islets of Langerhans. The α and β cells are electrically excitable and use electrical signals to couple changes in blood glucose concentration to stimulation or inhibition of hormone release. In both cell types, influx of extracellular Ca 2+ through voltage-gated Ca 2+ channels with resultant elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) triggers exocytosis of the hormone-containing secretory granules. Like other electrically excitable cells, both α and β cells contain several types of voltage-gated Ca 2+ channel (1, 2). Assigning physiological functions to the respective Ca 2+ channels is central to the understanding of electrical and secretory activities in these cells.Voltage-gated Ca 2+ channels are divided into 3 subfamilies: (a) L-type high voltage-activated (HVA) Ca 2+ channel family that comprises the Ca V 1.1, 1.2, 1.3, and 1.4 channels and is inhibited by dihydropyridines (DHPs) (1, 3, 4); (b) non-L-type HVA channels Ca V 2.1 (P/Q-type), 2.2 (N-type), and 2.3 (R-type) that are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA and SNX482, respectively (1, 4, 5); and (c) the low voltage-activated (LVA) T-type Ca 2+ channel family (Ca V 3.1, 3.2, and 3.3). The latter subtype differs electrophysiologically from the HVA Ca 2+ channels in opening transiently already upon modest depolarization (6, 7) and fulfilling important roles in pacemaker cells (8).

SUMMARY The TCF-1 and LEF-1 transcription factors are known to play critical roles in normal thymocyte development. Unexpectedly, we found that TCF-1-deficient (Tcf7−/−) mice developed aggressive T-cell malignancy, resembling human T-cell acute lymphoblastic leukemia (T-ALL). Surprisingly, LEF-1 was aberrantly upregulated in pre-malignant Tcf7−/− early thymocytes and lymphoma cells. We further demonstrated that TCF-1 directly repressed LEF-1 expression in early thymocytes and that conditional inactivation of Lef1 greatly delayed or prevented T-cell malignancy in Tcf7−/− mice. In human T-ALLs, an early thymic progenitor (ETP) subtype was associated with diminished TCF7 expression, and two of the ETP-ALL cases harbored TCF7 gene deletions. We also showed that TCF-1 and LEF-1 were dispensable for T-lineage commitment but instead were required for early thymocytes to mature beyond the CD4−CD8− stage. TCF-1 thus has dual roles, i.e., acting cooperatively with LEF-1 to promote thymocyte maturation while restraining LEF-1 expression to prevent malignant transformation of developing thymocytes.

Concerted activation of different voltage-gated Ca 2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca V 2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca V 2.3-knockout (Ca V 2.3 -/-) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca 2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca V 2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca 2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca V 2.3 -/-β cells. Ca V 2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca V 2.3 -/-mouse. We propose a specific role for Ca V 2.3 Ca 2+ channels in second-phase insulin release, that of mediating the Ca 2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation. IntroductionSystemic glucose tolerance is orchestrated by the regulated release of insulin and glucagon from the β and α cells of the pancreatic islets of Langerhans. The α and β cells are electrically excitable and use electrical signals to couple changes in blood glucose concentration to stimulation or inhibition of hormone release. In both cell types, influx of extracellular Ca 2+ through voltage-gated Ca 2+ channels with resultant elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) triggers exocytosis of the hormone-containing secretory granules. Like other electrically excitable cells, both α and β cells contain several types of voltage-gated Ca 2+ channel (1, 2). Assigning physiological functions to the respective Ca 2+ channels is central to the understanding of electrical and secretory activities in these cells.Voltage-gated Ca 2+ channels are divided into 3 subfamilies: (a) L-type high voltage-activated (HVA) Ca 2+ channel family that comprises the Ca V 1.1, 1.2, 1.3, and 1.4 channels and is inhibited by dihydropyridines (DHPs) (1, 3, 4); (b) non-L-type HVA channels Ca V 2.1 (P/Q-type), 2.2 (N-type), and 2.3 (R-type) that are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA and SNX482, respectively (1, 4, 5); and (c) the low voltage-activated (LVA) T-type Ca 2+ channel family (Ca V 3.1, 3.2, and 3.3). The latter subtype differs electrophysiologically from the HVA Ca 2+ channels in opening transiently already upon modest depolarization (6, 7) and fulfilling important roles in pacemaker cells (8).