Positional cloning of a type 2 diabetes quantitative trait locus; tomosyn-2, a negative regulator of insulin secretion.

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep(ob/ob) and C57BL/6 (B6) Lep(ob/ob) mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16(BT36-38)) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16(BT36-38) mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic ß-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion.

Monocytes in type 1 diabetes families exhibit high cytolytic activity and subset abundances that correlate with clinical progression.

Monocytes are immune regulators implicated in the pathogenesis of type 1 diabetes (T1D), an autoimmune disease that targets insulin-producing pancreatic ß cells. We determined that monocytes of recent onset (RO) T1D patients and their healthy siblings express proinflammatory/cytolytic transcriptomes and hypersecrete cytokines in response to lipopolysaccharide exposure compared to unrelated healthy controls (uHCs). Flow cytometry measured elevated circulating abundances of intermediate monocytes and >2-fold more CD14+CD16+HLADR+KLRD1+PRF1+ NK-like monocytes among patients with ROT1D compared to uHC. The intermediate to nonclassical monocyte ratio among ROT1D patients correlated with the decline in functional ß cell mass during the first 24 months after onset. Among sibling nonprogressors, temporal decreases were measured in the intermediate to nonclassical monocyte ratio and NK-like monocyte abundances; these changes coincided with increases in activated regulatory T cells. In contrast, these monocyte populations exhibited stability among T1D progressors. This study associates heightened monocyte proinflammatory/cytolytic activity with T1D susceptibility and progression and offers insight to the age-dependent decline in T1D susceptibility.

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of ß-cell mass.

Insufficient plasma insulin levels caused by deficits in both pancreatic ß-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed ß-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating ß-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in ß-cell proliferation is correlated with significantly increased ß-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of ß-cell decompensation: insufficient ß-cell function and mass.

Tcf19 is a novel islet factor necessary for proliferation and survival in the INS-1 ß-cell line.

Recently, a novel type 1 diabetes association locus was identified at human chromosome 6p31.3, and transcription factor 19 (TCF19) is a likely causal gene. Little is known about Tcf19, and we now show that it plays a role in both proliferation and apoptosis in insulinoma cells. Tcf19 is expressed in mouse and human islets, with increasing mRNA expression in nondiabetic obesity. The expression of Tcf19 is correlated with ß-cell mass expansion, suggesting that it may be a transcriptional regulator of ß-cell mass. Increasing proliferation and decreasing apoptotic cell death are two strategies to increase pancreatic ß-cell mass and prevent or delay diabetes. siRNA-mediated knockdown of Tcf19 in the INS-1 insulinoma cell line, a ß-cell model, results in a decrease in proliferation and an increase in apoptosis. There was a significant reduction in the expression of numerous cell cycle genes from the late G1 phase through the M phase, and cells were arrested at the G1/S checkpoint. We also observed increased apoptosis and susceptibility to endoplasmic reticulum (ER) stress after Tcf19 knockdown. There was a reduction in expression of genes important for the maintenance of ER homeostasis (Bip, p58(IPK), Edem1, and calreticulin) and an increase in proapoptotic genes (Bim, Bid, Nix, Gadd34, and Pdia2). Therefore, Tcf19 is necessary for both proliferation and survival and is a novel regulator of these pathways.

Albumin nanoparticle containing a PI3Kγ inhibitor and paclitaxel in combination with α-PD1 induces tumor remission of breast cancer in mice.

Immunomodulators that remodel the tumor immunosuppressive microenvironment have been combined with anti-programmed death 1 (α-PD1) or anti-programmed death ligand 1 (α-PDL1) immunotherapy but have shown limited success in clinical trials. However, therapeutic strategies to modulate the immunosuppressive microenvironment of lymph nodes have been largely overlooked. Here, we designed an albumin nanoparticle, Nano-PI, containing the immunomodulators PI3Kγ inhibitor (IPI-549) and paclitaxel (PTX). We treated two breast cancer mouse models with Nano-PI in combination with α-PD1, which remodeled the tumor microenvironment in both lymph nodes and tumors. This combination achieved long-term tumor remission in mouse models and eliminated lung metastases. PTX combined with IPI-549 enabled the formation of a stable nanoparticle and enhanced the repolarization of M2 to M1 macrophages. Nano-PI not only enhanced the delivery of both immunomodulators to lymph nodes and tumors but also improved the drug accumulation in the macrophages of these two tissues. Immune cell profiling revealed that the combination of Nano-PI with α-PD1 remodeled the immune microenvironment by polarizing M2 to M1 macrophages, increasing CD4+ and CD8+ T cells, B cells, and dendritic cells, decreasing regulatory T cells, and preventing T cell exhaustion. Our data suggest that Nano-PI in combination with α-PD1 modulates the immune microenvironment in both lymph nodes and tumors to achieve long-term remission in mice with metastatic breast cancer, and represents a promising candidate for future clinical trials.

Human Islet Expression Levels of Prostaglandin E2 Synthetic Enzymes, But Not Prostaglandin EP3 Receptor, Are Positively Correlated with Markers of ß-Cell Function and Mass in Nondiabetic Obesity.

Elevated islet production of prostaglandin E2 (PGE2), an arachidonic acid metabolite, and expression of prostaglandin E2 receptor subtype EP3 (EP3) are well-known contributors to the ß-cell dysfunction of type 2 diabetes (T2D). Yet, many of the same pathophysiological conditions exist in obesity, and little is known about how the PGE2 production and signaling pathway influences nondiabetic ß-cell function. In this work, plasma arachidonic acid and PGE2 metabolite levels were quantified in a cohort of nondiabetic and T2D human subjects to identify their relationship with glycemic control, obesity, and systemic inflammation. In order to link these findings to processes happening at the islet level, cadaveric human islets were subject to gene expression and functional assays. Interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA levels, but not those of EP3, positively correlated with donor body mass index (BMI). IL-6 expression also strongly correlated with the expression of COX-2 and other PGE2 synthetic pathway genes. Insulin secretion assays using an EP3-specific antagonist confirmed functionally relevant upregulation of PGE2 production. Yet, islets from obese donors were not dysfunctional, secreting just as much insulin in basal and stimulatory conditions as those from nonobese donors as a percent of content. Islet insulin content, on the other hand, was increased with both donor BMI and islet COX-2 expression, while EP3 expression was unaffected. We conclude that upregulated islet PGE2 production may be part of the ß-cell adaption response to obesity and insulin resistance that only becomes dysfunctional when both ligand and receptor are highly expressed in T2D.

The Inhibitory G Protein α-Subunit, Gαz, Promotes Type 1 Diabetes-Like Pathophysiology in NOD Mice.

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes ß-cell death and inhibits ß-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional ß-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive ß-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive ß-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, ß-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a ß-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in ß-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of ß-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

Pancreatic ß-Cells From Mice Offset Age-Associated Mitochondrial Deficiency With Reduced KATP Channel Activity.

Aging is accompanied by impaired glucose homeostasis and an increased risk of type 2 diabetes, culminating in the failure of insulin secretion from pancreatic ß-cells. To investigate the effects of age on ß-cell metabolism, we established a novel assay to directly image islet metabolism with NAD(P)H fluorescence lifetime imaging (FLIM). We determined that impaired mitochondrial activity underlies an age-dependent loss of insulin secretion in human islets. NAD(P)H FLIM revealed a comparable decline in mitochondrial function in the pancreatic islets of aged mice (≥24 months), the result of 52% and 57% defects in flux through complex I and II, respectively, of the electron transport chain. However, insulin secretion and glucose tolerance are preserved in aged mouse islets by the heightened metabolic sensitivity of the ß-cell triggering pathway, an adaptation clearly encoded in the metabolic and Ca(2+) oscillations that trigger insulin release (Ca(2+) plateau fraction: young 0.211 ± 0.006, aged 0.380 ± 0.007, P < 0.0001). This enhanced sensitivity is driven by a reduction in KATP channel conductance (diazoxide: young 5.1 ± 0.2 nS; aged 3.5 ± 0.5 nS, P < 0.01), resulting in an ∼2.8 mmol/L left shift in the ß-cell glucose threshold. The results demonstrate how mice but not humans are able to successfully compensate for age-associated metabolic dysfunction by adjusting ß-cell glucose sensitivity and highlight an essential mechanism for ensuring the maintenance of insulin secretion.

A single-islet microplate assay to measure mouse and human islet insulin secretion.

One complication to comparing ß-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.

A method for mouse pancreatic islet isolation and intracellular cAMP determination.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The ß-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the ß-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on ß-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased ß-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3(H)]-thymidine incorporation, protein abundance, and mRNA expression.

Prostaglandin E2 receptor, EP3, is induced in diabetic islets and negatively regulates glucose- and hormone-stimulated insulin secretion.

BTBR mice develop severe diabetes in response to genetically induced obesity due to a failure of the ß-cells to compensate for peripheral insulin resistance. In analyzing BTBR islet gene expression patterns, we observed that Pgter3, the gene for the prostaglandin E receptor 3 (EP3), was upregulated with diabetes. The EP3 receptor is stimulated by prostaglandin E2 (PGE2) and couples to G-proteins of the Gi subfamily to decrease intracellular cAMP, blunting glucose-stimulated insulin secretion (GSIS). Also upregulated were several genes involved in the synthesis of PGE2. We hypothesized that increased signaling through EP3 might be coincident with the development of diabetes and contribute to ß-cell dysfunction. We confirmed that the PGE2-to-EP3 signaling pathway was active in islets from confirmed diabetic BTBR mice and human cadaveric donors, with increased EP3 expression, PGE2 production, and function of EP3 agonists and antagonists to modulate cAMP production and GSIS. We also analyzed the impact of EP3 receptor activation on signaling through the glucagon-like peptide (GLP)-1 receptor. We demonstrated that EP3 agonists antagonize GLP-1 signaling, decreasing the maximal effect that GLP-1 can elicit on cAMP production and GSIS. Taken together, our results identify EP3 as a new therapeutic target for ß-cell dysfunction in T2D.