Ainhoa Garcia

Deletion of miRNA processing enzyme Dicer in POMC-expressing cells leads to pituitary dysfunction, neurodegeneration and development of obesity.

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin-proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity.

Reduced α-MSH Underlies Hypothalamic ER-Stress-Induced Hepatic Gluconeogenesis

Alterations in ER homeostasis have been implicated in the pathophysiology of obesity and type-2 diabetes (T2D). Acute ER stress induction in the hypothalamus produces glucose metabolism perturbations. However, the neurobiological basis linking hypothalamic ER stress with abnormal glucose metabolism remains unknown. Here, we report that genetic and induced models of hypothalamic ER stress are associated with alterations in systemic glucose homeostasis due to increased gluconeogenesis (GNG) independent of body weight changes. Defective alpha melanocyte-stimulating hormone (α-MSH) production underlies this metabolic phenotype, as pharmacological strategies aimed at rescuing hypothalamic α-MSH content reversed this phenotype at metabolic and molecular level. Collectively, our results posit defective α-MSH processing as a fundamental mediator of enhanced GNG in the context of hypothalamic ER stress and establish α-MSH deficiency in proopiomelanocortin (POMC) neurons as a potential contributor to the pathophysiology of T2D.

Human Serum Versus Human Serum Albumin Supplementation in Human Islet Pretransplantation Culture: In Vitro and In Vivo Assessment

There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS-and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted.

Tungstate promotes β-cell survival in Irs2−/− mice

Pancreatic β-cells play a central role in type 2 diabetes (T2D) development, which is characterized by the progressive decline of the functional β-cell mass that is associated mainly with increased β-cell apoptosis. Thus, understanding how to enhance survival of β-cells is key for the management of T2D. The insulin receptor substrate-2 (IRS-2) protein is pivotal in mediating the insulin/IGF signaling pathway in β-cells. In fact, IRS-2 is critically required for β-cell compensation in conditions of increased insulin demand and for β-cell survival. Tungstate is a powerful antidiabetic agent that has been shown to promote β-cell recovery in toxin-induced diabetic rodent models. In this study, we investigated whether tungstate could prevent the onset of diabetes in a scenario of dysregulated insulin/IGF signaling and massive β-cell death. To this end, we treated mice deficient in IRS2 (Irs2(-/-)), which exhibit severe β-cell loss, with tungstate for 3 wk. Tungstate normalized glucose tolerance in Irs2(-/-) mice in correlation with increased β-cell mass, increased β-cell replication, and a striking threefold reduction in β-cell apoptosis. Islets from treated Irs2(-/-) exhibited increased phosphorylated Erk1/2. Interestingly, tungstate repressed apoptosis-related genes in Irs2(-/-) islets in vitro, and ERK1/2 blockade abolished some of these effects. Gene expression profiling showed evidence of a broad impact of tungstate on cell death pathways in islets from Irs2(-/-) mice, consistent with reduced apoptotic rates. Our results support the finding that β-cell death can be arrested in the absence of IRS2 and that therapies aimed at reversing β-cell mass decline are potential strategies to prevent the progression to T2D.

Mycobacterium tuberculosis multi‐drug‐resistant strain M induces IL‐17+IFNγ– CD4+ T cell expansion through an IL‐23 and TGF‐β‐dependent mechanism in patients with MDR‐TB tuberculosis

We have reported previously that T cells from patients with multi‐drug‐resistant tuberculosis (MDR‐TB) express high levels of interleukin (IL)‐17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR‐TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD+ HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL‐1β and IL‐6 are crucial for the H37Rv and M‐induced expansion of IL‐17+interferon (IFN)‐γ– and IL‐17+IFN‐γ+ in CD4+ T cells from MDR‐TB and PPD+ HD. IL‐23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL‐23 is responsible for M. tuberculosis‐induced IL‐17 and IFN‐γ expression in CD4+ T cells from PPD+ HD whereas, together with transforming growth factor (TGF‐β), it promotes IL‐17+IFN‐γ– expansion in MDR‐TB. In fact, spontaneous and M. tuberculosis‐induced TGF‐β secretion is increased in cells from MDR‐TB, the M strain being the highest inducer. Interestingly, Toll‐like receptor (TLR)‐2 signalling mediates the expansion of IL‐17+IFN‐γ– cells and the enhancement of latency‐associated protein (LAP) expression in CD14+ and CD4+ T cells from MDR‐TB, which suggests that the M strain promotes IL‐17+IFN‐γ– T cells through a strong TLR‐2‐dependent TGF‐β production by antigen‐presenting cells and CD4+ T cells. Finally, CD4+ T cells from MDR‐TB patients infected with MDR Haarlem strains show higher IL‐17+IFN‐γ– and lower IL‐17+IFN‐γ+ levels than LAM‐infected patients. The present findings deepen our understanding of the role of IL‐17 in MDR‐TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex‐vivo Th17 response.

Wnt9a deficiency discloses a repressive role of Tcf7l2 on endocrine differentiation in the embryonic pancreas

Transcriptional and signaling networks establish complex cross-regulatory interactions that drive cellular differentiation during development. Using microarrays we identified the gene encoding the ligand Wnt9a as a candidate target of Neurogenin3, a basic helix-loop-helix transcription factor that functions as a master regulator of pancreatic endocrine differentiation. Here we show that Wnt9a is expressed in the embryonic pancreas and that its deficiency enhances activation of the endocrine transcriptional program and increases the number of endocrine cells at birth. We identify the gene encoding the endocrine transcription factor Nkx2-2 as one of the most upregulated genes in Wnt9a-ablated pancreases and associate its activation to reduced expression of the Wnt effector Tcf7l2. Accordingly, in vitro studies confirm that Tcf7l2 represses activation of Nkx2-2 by Neurogenin3 and inhibits Nkx2-2 expression in differentiated β-cells. Further, we report that Tcf7l2 protein levels decline upon initiation of endocrine differentiation in vivo, disclosing the downregulation of this factor in the developing endocrine compartment. These findings highlight the notion that modulation of signalling cues by lineage-promoting factors is pivotal for controlling differentiation programs.

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,

Late-stage differentiation of embryonic pancreatic β-cells requires Jarid2.

Jarid2 is a component of the Polycomb Repressor complex 2 (PRC2), which is responsible for genome-wide H3K27me3 deposition, in embryonic stem cells. However, Jarid2 has also been shown to exert pleiotropic PRC2-independent actions during embryogenesis. Here, we have investigated the role of Jarid2 during pancreas development. Conditional ablation of Jarid2 in pancreatic progenitors results in reduced endocrine cell area at birth due to impaired endocrine cell differentiation and reduced prenatal proliferation. Inactivation of Jarid2 in endocrine progenitors demonstrates that Jarid2 functions after endocrine specification. Furthermore, genome-wide expression analysis reveals that Jarid2 is required for the complete activation of the insulin-producing β-cell differentiation program. Jarid2-deficient pancreases exhibit impaired deposition of RNAPII-Ser5P, the initiating form of RNAPII, but no changes in H3K27me3, at the promoters of affected endocrine genes. Thus, our study identifies Jarid2 as a fine-tuner of gene expression during late stages of pancreatic endocrine cell development. These findings are relevant for generation of transplantable stem cell-derived β-cells.