Conchi Mora

Insulin-Induced Redistribution of GLUT4 Glucose Carriers in the Muscle Fiber: In Search of GLUT4 Trafficking Pathways

Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulinresistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLTJT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarco-lemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells.

Human DQ8 Can Substitute for Murine I-Ag7 in the Selection of Diabetogenic T Cells Restricted to I-Ag71

The strong association of type 1 diabetes with specific MHC class II genes, such as I-Ag7 in nonobese diabetic mice and HLA-DQ8 in humans, suggests that MHC class II molecules play an important role in the development of the disease. To test whether human DQ8 molecules could cross the species barrier and functionally replace their murine homolog I-Ag7, we generated DQ8/BDC2.5 transgenic mice. We have shown that BDC2.5 transgenic T cells are selected on DQ8 in the thymus and cause diabetes in a manner similar to that seen when the T cells are selected on H2g7. Splenocytes from DQ8/BDC2.5 mice also showed reactivity toward islets in vitro as seen in H-2g7/BDC2.5 mice. We conclude that DQ8 molecules not only share structural similarity with the murine homolog I-Ag7, but also can cross the species barrier and functionally replace I-Ag7 molecules to stimulate diabetogenic T cells and produce diabetes.

In Vivo Detection of Peripherin-Specific Autoreactive B Cells during Type 1 Diabetes Pathogenesis

Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. In this study, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target Ag of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cells during disease progression. Before extended insulitis was established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not prediabetic mice. Isotype-switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes is established. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis.

Cyclin D3 promotes pancreatic β-cell fitness and viability in a cell cycle-independent manner and is targeted in autoimmune diabetes

Significance Autoimmune diabetes is caused by the lymphocyte-mediated destruction of the pancreatic insulin-producing β cells. The inflammatory niche surrounding β cells prior to diabetes onset provokes death-inducing molecular changes in these cells. Unknown molecular pathways related to β-cell viability that are altered by inflammation in vivo, and therefore potential therapeutic targets, remain to be identified, because most of the previous studies took in vitro approaches. Here, we report that cyclin D3, classically related to cell proliferation, is targeted in autoimmune diabetes and exerts a protective role on β cells by promoting their survival and fitness without affecting proliferation. These findings unveil a dual, cell cycle-independent role of cyclin D3 with high potential in the areas of autoimmunity and metabolism. Type 1 diabetes is an autoimmune condition caused by the lymphocyte-mediated destruction of the insulin-producing β cells in pancreatic islets. We aimed to identify final molecular entities targeted by the autoimmune assault on pancreatic β cells that are causally related to β cell viability. Here, we show that cyclin D3 is targeted by the autoimmune attack on pancreatic β cells in vivo. Cyclin D3 is down-regulated in a dose-dependent manner in β cells by leukocyte infiltration into the islets of the nonobese diabetic (NOD) type 1 diabetes-prone mouse model. Furthermore, we established a direct in vivo causal link between cyclin D3 expression levels and β-cell fitness and viability in the NOD mice. We found that changes in cyclin D3 expression levels in vivo altered the β-cell apoptosis rates, β-cell area homeostasis, and β-cell sensitivity to glucose without affecting β-cell proliferation in the NOD mice. Cyclin D3-deficient NOD mice exhibited exacerbated diabetes and impaired glucose responsiveness; conversely, transgenic NOD mice overexpressing cyclin D3 in β cells exhibited mild diabetes and improved glucose responsiveness. Overexpression of cyclin D3 in β cells of cyclin D3-deficient mice rescued them from the exacerbated diabetes observed in transgene-negative littermates. Moreover, cyclin D3 overexpression protected the NOD-derived insulinoma NIT-1 cell line from cytokine-induced apoptosis. Here, for the first time to our knowledge, cyclin D3 is identified as a key molecule targeted by autoimmunity that plays a nonredundant, protective, and cell cycle-independent role in β cells against inflammation-induced apoptosis and confers metabolic fitness to these cells.

Targeting of a T Cell Agonist Peptide to Lysosomes by DNA Vaccination Induces Tolerance in the Nonobese Diabetic Mouse

CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides to the lysosomal compartment on a monospecific T cell population termed 2.5mi+ T cells that shares reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse. MHC class II tetramer analysis showed that repeated administrations were necessary to expand 2.5mi+ T cells in vivo. This expansion was independent of Ag presentation by B cells. A single peptide epitope was sufficient to induce protection against T1D, which was not due to Ag-specific T cell anergy. Typical Th2 cytokines such as IL-10 or IL-4 were undetectable in 2.5mi+ T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi+ T cell population produced IFN-γ similar to 2.5mi+ T cells from naive mice. Protection against T1D by DNA treatment was completely lost in NOD.CD28−/− mice which are largely deficient of natural regulatory T cells (Treg). Although Ag-specific Foxp3+ Treg did not expand in response to DNA treatment, diabetes onset was delayed in Treg-reconstituted and DNA-treated NOD.SCID mice. These observations provide evidence for a Treg-mediated protective mechanism that is independent of the expansion or de novo generation of Ag-specific Treg.

Cholera toxin subunit B peptide fusion proteins reveal impaired oral tolerance induction in diabetes‐prone but not in diabetes‐resistant mice

The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag‐specific CD4+ T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4+ T‐cell epitopes, the BDC‐2.5 T‐cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4+ T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB‐2.5mi activated 2.5mi+ T cells and following intragastric delivery generated Ag‐specific Foxp3+ Treg and Th2 cells. While 2.5mi+ and GAD‐specific T cells were tolerized in diabetes‐resistant NODxB6.Foxp3EGFP F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB‐fused epitopes. In contrast to NODxB6.Foxp3EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi+ T‐cell activation and the sequestration of these cells to the effector‐memory pool. Oral treatment of NOD mice with CTB‐2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.

LoVo colon cancer cells resistant to oxaliplatin overexpress c-MET and VEGFR-1 and respond to VEGF with dephosphorylation of c-MET.

Oxaliplatin‐resistant LoVo colon cancer cells overexpressing c‐MET and VEGFR‐1 were selected to study several signaling pathways involved in chemoresistance, as well as the effect of increasing amounts of VEGF in the regulation of c‐MET. In comparison with chemosensitive LoVo colon cancer cells, oxaliplatin‐resistant cells (LoVoR) overexpress and phosphorylate c‐MET, upregulate the expression of transmembrane and soluble VEGFR‐1 and, unexpectedly, downregulate VEGF. In addition, LoVoR cells activate other transduction pathways involved in chemoresistance such as Akt, β‐catenin‐TCF4 and E‐cadherin. While c‐MET is phosphorylated in LoVoR cells expressing low levels of VEGF, c‐MET phosphorylation decreases when recombinant VEGF is added into the culture medium. Inhibition of c‐MET by VEGF is mediated by VEGFR‐1, since phosphorylation of c‐MET in the presence of VEGF is restored after silencing VEGFR‐1. Dephosphorylation of c‐MET by VEGF suggests that tumors coexpressing VEGFR‐1 and c‐MET may activate c‐MET as a result of anti‐VEGF therapy.

B-cell anergy induces a Th17 shift in a novel B lymphocyte transgenic NOD mouse model, the 116C-NOD mouse.

Autoreactive B lymphocytes play a key role as APCs in diaebetogenesis. However, it remains unclear whether B‐cell tolerance is compromised in NOD mice. Here, we describe a new B lymphocyte transgenic NOD mouse model, the 116C‐NOD mouse, where the transgenes derive from an islet‐infiltrating B lymphocyte of a (8.3‐NODxNOR) F1 mouse. The 116C‐NOD mouse produces clonal B lymphocytes with pancreatic islet beta cell specificity. The incidence of T1D in 116C‐NOD mice is decreased in both genders when compared with NOD mice. Moreover, several immune selection mechanisms (including clonal deletion and anergy) acting on the development, phenotype, and function of autoreactive B lymphocytes during T1D development have been identified in the 116C‐NOD mouse. Surprisingly, a more accurate analysis revealed that, despite their anergic phenotype, 116C B cells express some costimulatory molecules after activation, and induce a T‐cell shift toward a Th17 phenotype. Furthermore, this shift on T lymphocytes seems to occur not only when both T and B cells contact, but also when helper T (Th) lineage is established. The 116C‐NOD mouse model could be useful to elucidate the mechanisms involved in the generation of Th‐cell lineages.

Treatment of T1D via optimized expansion of antigen-specific Tregs induced by IL-2/anti-IL-2 monoclonal antibody complexes and peptide/MHC tetramers

Type 1 diabetes can be overcome by regulatory T cells (Treg) in NOD mice yet an efficient method to generate and maintain antigen-specific Treg is difficult to come by. Here, we devised a combination therapy of peptide/MHC tetramers and IL-2/anti-IL-2 monoclonal antibody complexes to generate antigen-specific Treg and maintain them over extended time periods. We first optimized treatment protocols conceived to obtain an improved islet-specific Treg/effector T cell ratio that led to the in vivo expansion and activation of these Treg as well as to an improved suppressor function. Optimized protocols were applied to treatment for testing diabetes prevention in NOD mice as well as in an accelerated T cell transfer model of T1D. The combined treatment led to robust protection against diabetes, and in the NOD model, to a close to complete prevention of insulitis. Treatment was accompanied with increased secretion of IL-10, detectable in total splenocytes and in Foxp3− CD4 T cells. Our data suggest that a dual protection mechanism takes place by the collaboration of Foxp3+ and Foxp3− regulatory cells. We conclude that antigen-specific Treg are an important target to improve current clinical interventions against this disease.

The sole presence of CDK4 is not a solid criterion for discriminating between tumor and healthy pancreatic tissues

Dear Editor, Cyclin-dependent kinase 4 (CDK4) is a key protein in G1 transition during cell-cycle progression [1]. Two different groups have postulated that CDK4 may be considered a pancreatic tumor marker. These groups have not detected CDK4 expression in healthy pancreas, but they have detected CDK4 in pancreatic endocrine tumors [2], intraepithelial neoplasia and ductal adenocarcinoma of the pancreas [3]. These results clearly contrast with those obtained from murine models, in which CDK4 is the main G1 transition kinase in the pancreatic tissue. CDK4-null mice exhibit severe beta-cell mass hypoplasia, whereas islet CDK4 hyperactivity induces hyperplasia without causing hypoglycemia [4]. The explanation for this divergence was discussed at a symposium [5], where cyclin-dependent kinase 6 (CDK6) was proposed as the protein responsible for regulating G1 transition in the healthy pancreas of humans because only CDK6, not CDK4, has been detected in healthy pancreases. However, in a subsequent study, these authors suggested that healthy isolated human pancreatic islets express CDK6 and CDK4 [6]. To assess the usefulness of CDK4 as a biomarker of pancreatic cancer, we extensively examined its presence using several techniques in normal pancreas (n=24) and islets (n=11), pancreatic adenocarcinomas (n=125) and pancreatic neuroendocrine tumors (n=5). The detailed material and methods in the supplementary information are available at https://sites.google.com/site/conchimoralabs/cdk4-pancreas-supplementary-information. To observe the presence of CDK4 using immunohistochemistry, we used two similar antibodies against CDK4 (H-22 and C-22; Santa Cruz Technology, Santa Cruz, CA, USA), which were raised against the human or mouse C-terminal portion of CDK4, respectively. It is noteworthy that one of the research groups mentioned above failed to detect CDK4 by immunohistochemistry and western blot analysis in healthy human pancreas using the H-22 antibody [2]. We obtained similar results with both antibodies (see representative images in Fig. 1, A-H) and observed that 58% of the healthy samples (14 of 24), 67% of the adenocarcinomas (84 of 125) and 80% of the neuroendocrine tumors (4 of 5) were positive for CDK4 staining (detailed results are available in the supplementary information). To ensure staining specificity, we performed several quality controls. First, some human pancreatic sections were stained only with the secondary antibodies, which did not produce any staining signals. Second, Cdk4 staining of murine pancreatic sections showed a clear nuclear staining in the endocrine and exocrine pancreatic tissue. Third, pancreatic tissue sections of Cdk4-deficient mice were immunonegative for Cdk4 after the sections were incubated with the anti-Cdk4 antibody. Finally, CDK4 staining in human samples that were blocked with the CDK4-blocking peptide (Santa Cruz Technology) showed no signal. After performing all of these controls (representative images are available in the supplementary information), we concluded that the staining observed using the anti-CDK4 antibody in human pancreatic sections was specific. Figure 1 Top Panels (A-H). Two representative sections of healthy human pancreas from the UTIP healthy series (A-H) immunostained for CDK4 (A, E) and co-stained with insulin (B, F) and the nuclear marker Hoechst (Sigma) (C, G) for nuclear detection. Each row corresponds, ... The lack of homogeneity in healthy pancreatic tissue -only 58% of the human healthy pancreatic samples showed CDK4 staining- may be caused by the expected heterogeneity within the human species or differences in processing of samples before performing the immunohistochemistry technique (time before organ extraction, fixation time, etc.), which remains to be explored in depth. We evaluated the presence of CDK4 in human healthy islets using real-time PCR and western blot analysis (Fig. 1, I-L). The presence of CDK4 was detected using western blot analysis with four different anti-CDK4 antibodies: DCS-31 (Sigma, St. Louis, MO, USA), DCS-156 (Becton Dickinson, Franklin Lakes, NJ, USA), C-22 and H-22 (Santa Cruz Technology). To ensure the specificity of the results, we performed several quality controls. First, we observed a clear western blot band using mouse pancreas lysates. Second, this band was not present in the lysates of the Cdk4 knockout mouse pancreas. Third, the incubation of the antibody with the corresponding blocking peptide did not render any signal in human healthy islets (representative images are available in the supplementary information). The role of CDK4 in cell-cycle progression has been clearly demonstrated [1]. Therefore, we co-stained for CDK4 and the proliferation marker Ki-67. We found no correlation between Ki-67 and CDK4 staining. These results suggest that the sole presence of CDK4 is not indicative of proliferation (Fig. 1, M-X), All of these results indicate that CDK4 is not useful as a tumor marker. We showed that many healthy pancreatic samples were positive for this protein using different techniques. Moreover, the in-depth analysis of the immunohistochemistry results from the SEER array (Surveillance Epidemiology and End Results (SEER) Residual Tissue Repository tissue array (Bethesda, MD, USA) [7]) reveal that, after adjusting for demographic and clinical attributes, the survival analysis for 50 cases of pancreatic ductal adenocarcinoma-resection tumors (41 cases with nuclear CDK4 staining versus 9 cases without nuclear CDK4 staining), showed a marginally improved survival time for the cases that exhibited nuclear CDK4 staining versus those that did not (log-rank test, p=0.016; hazard ratio, 2.2; 95% confidence interval, 1.0–4.8). In conclusion, our results suggest that the presence of CDK4 alone cannot be used as a pancreatic tumor marker to distinguish between normal and tumor pancreas. In addition, we hypothesize that the role of CDK4 in pancreatic tissue may extend beyond cell-cycle progression, as CDK4 is also involved in other processes as the regulation of insulin secretion in beta-cells [8]. Yours sincerely,