Nadia Cobo-Vuilleumier

Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors

The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker.

Opposite clinical phenotypes of glucokinase disease: Description of a novel activating mutation and contiguous inactivating mutations in human glucokinase (GCK) gene

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

The liver receptor homolog-1 (LRH-1) is expressed in human islets and protects β-cells against stress-induced apoptosis

Liver receptor homolog (LRH-1) is an orphan nuclear receptor (NR5A2) that regulates cholesterol homeostasis and cell plasticity in endodermal-derived tissues. Estrogen increases LRH-1 expression conveying cell protection and proliferation. Independently, estrogen also protects isolated human islets against cytokine-induced apoptosis. Herein, we demonstrate that LRH-1 is expressed in islets, including β-cells, and that transcript levels are modulated by 17β-estradiol through the estrogen receptor (ER)α but not ERβ signaling pathway. Repression of LRH-1 by siRNA abrogated the protective effect conveyed by estrogen on rat islets against cytokines. Adenoviral-mediated overexpression of LRH-1 in human islets did not alter proliferation but conferred protection against cytokines and streptozotocin-induced apoptosis. Expression levels of the cell cycle genes cyclin D1 and cyclin E1 as well as the antiapoptotic gene bcl-xl were unaltered in LRH-1 expressing islets. In contrast, the steroidogenic enzymes CYP11A1 and CYP11B1 involved in glucocorticoid biosynthesis were both stimulated in transduced islets. In parallel, graded overexpression of LRH-1 dose-dependently impaired glucose-induced insulin secretion. Our results demonstrate the crucial role of the estrogen target gene nr5a2 in protecting human islets against-stressed-induced apoptosis. We postulate that this effect is mediated through increased glucocorticoid production that blunts the pro-inflammatory response of islets.

Islet β-Cell Mass Preservation and Regeneration in Diabetes Mellitus: Four Factors with Potential Therapeutic Interest

Islet β-cell replacement and regeneration are two promising approaches for the treatment of Type 1 Diabetes Mellitus. Indeed, the success of islet transplantation in normalizing blood glucose in diabetic patients has provided the proof of principle that cell replacement can be employed as a safe and efficacious treatment. Nonetheless, shortage of organ donors has hampered expansion of this approach. Alternative sources of insulin-producing cells are mandatory to fill this gap. Although great advances have been achieved in generating surrogate β-cells from stem cells, current protocols have yet to produce functionally mature insulin-secreting cells. Recently, the concept of islet regeneration in which new β-cells are formed from either residual β-cell proliferation or transdifferentiation of other endocrine islet cells has gained much interest as an attractive therapeutic alternative to restore β-cell mass. Complementary approaches to cell replacement and regeneration could aim at enhancing β-cell survival and function. Herein, we discuss the value of Hepatocyte Growth Factor (HGF), Glucose-Dependent Insulinotropic Peptide (GIP), Paired box gene 4 (Pax4) and Liver Receptor Homolog-1 (LRH-1) as key players for β-cell replacement and regeneration therapies. These factors convey β-cell protection and enhanced function as well as facilitating proliferation and transdifferentiation of other pancreatic cell types to β-cells, under stressful conditions.

Pax8 detection in well-differentiated pancreatic endocrine tumors: how reliable is it?

To the Editor: One of the most challenging areas in pancreatic endocrine tumor (PET) biology is the identification of specific biomarkers that can morphologically and histochemically distinguish well-differentiated PETs from other types of neuroendocrine tumors (NETs). Novel markers could improve diagnosis accuracy and could consequently lead to a more appropriate and tailored treatment and therefore to a better prognosis. This is highly relevant from the clinical point of view as at least 10% of patients with NETs have a widespread disease of unknown primary origin, and available antineoplastic agents are fundamentally effective only on tumors of pancreatic origin. Unfortunately, current markers including synaptophysin and chromogranin A are unable to distinguish whether a particular NET originated initially from the endocrine pancreas or from other sites such as the gastrointestinal tract or the bronchopulmonary system. In this regard, a recent study published in the American Journal of Surgical Pathology has identified the transcription factor Pax8 as a potentially new biomarker capable of performing such a task. Although previously not detected in the pancreas, Long and colleagues reported strong Pax8 immunostaining in normal human islets and have suggested that this transcription factor could be a specific marker of mature pancreatic endocrine cells and could even be implicated in terminal differentiation of islet cells during development. This premise was corroborated by a second American Journal of Surgical Pathology publication confirming the expression of Pax8 in healthy human islets. The discovery of Pax8 expression in the endocrine pancreas led Long and colleagues to assess whether Pax8 was expressed in PETs and in other well-differentiated NETs and to evaluate its potential value as a specific biomarker. They showed that a significant percentage of primary welldifferentiated PETs displayed strong Pax8 staining, whereas ileal and pulmonary carcinoid tumors were negative. Furthermore, liver metastases of PET origin were often Pax8 positive. The investigators concluded that Pax8 could be a useful marker to determine the primary origin site of metastatic well-differentiated PETs lodged within the liver. Interestingly, an independent study substantiated these findings and proposed a flowchart in which Pax8 was included in a panel of immunohistochemical stainings to determine the site of origin of metastatic well-differentiated NET in the liver. Unfortunately, we would like to communicate to the American Journal of Surgical Pathology readership that the conclusion of these studies is potentially inaccurate and that Pax8 proteins are not detectable in either healthy islets or PETs. Indeed, subsequent to the publication by Long et al we embarked on a study to determine whether Pax8 could also be expressed during pancreatic islet development, thereby pinpointing to a yet unknown function of this transcription factor in islet physiology. To this end, we analyzed Pax8 expression during mouse pancreatic development and in adult islets by immunohistochemistry using the same Pax8 polyclonal antibody (ProteinTech Group Inc., Chicago, IL; Cat. No. 10336-1AP) that Long et al used in their study. It is noteworthy that this polyclonal antibody was also used for most, if not all, clinical studies that demonstrated Pax8 staining in PETs. Consistent with these published reports we were successful in detecting Pax8 immunostaining in mature mouse and human islets and in the developing mouse pancreas. However, in an attempt to complement our immunohistochemical data with transcript expression profile, we unexpectedly found that Pax8 mRNA levels in both human and mouse islets were low to undetectable. These findings led us to question the specificity of the Pax8 polyclonal antibody. Indeed, as the 9 Pax members share a high degree of homology at the N-terminal paired domain, the region included in the peptide use for originating the antibody, a potential cross-reactivity of the antibody to another Pax member could be envisaged. This premise was indeed confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and by immunohistochemistry. To circumvent this caveat, a novel Pax8 monoclonal antibody from Abcam (No. ab53490) generated against the more variable C-terminal region of Pax8 was used to reevaluate the expression pattern of Pax8 during development and in islets. This antibody recognizes the 2 predominant isoforms of Pax8 expressed in most tissues and tumors. Consistent with our QT-RTPCR data, neither the developing pancreas nor mature islets exhibited staining with this novel Pax8 monoclonal antibody as compared with the polyclonal antibody. These results led us to reassess whether Pax8 was truly present in PETs. To this end, serial sections of PETs were immunostained with Pax8 antibodies (polyclonal and monoclonal) and with anti-Pax6 sera (Fig. 1). Immunohistochemical analysis revealed positive staining both in nonneoplastic islets and in tumor areas of pancreas sections when the Pax8 polyclonal antibody was used, whereas no staining was revealed with the Pax8 monoclonal antibody (Figs. 1A, B). An immunostaining pattern similar to that of the Pax8 polyclonal antibody was discerned with the Pax6 antibody, substantiating cross-reactivity of the commonly used Pax8 polyclonal antibody with Pax6 and possibly with other members of the Pax family (Fig. 1C). Thus, in contrast to the study by Long et al, we LETTER TO THE EDITOR

PAX4 Defines an Expandable β-Cell Subpopulation in the Adult Pancreatic Islet

PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets.

The Diabetes-Linked Transcription Factor PAX4: From Gene to Functional Consequences

Paired box 4 (PAX4) is a key factor in the generation of insulin producing β-cells during embryonic development. In adult islets, PAX4 expression is sequestered to a subset of β-cells that are prone to proliferation and more resistant to stress-induced apoptosis. The importance of this transcription factor for adequate pancreatic islets functionality has been manifested by the association of mutations in PAX4 with the development of diabetes, independently of its etiology. Overexpression of this factor in adult islets stimulates β-cell proliferation and increases their resistance to apoptosis. Additionally, in an experimental model of autoimmune diabetes, a novel immunomodulatory function for this factor has been suggested. Altogether these data pinpoint at PAX4 as an important target for novel regenerative therapies for diabetes treatment, aiming at the preservation of the remaining β-cells in parallel to the stimulation of their proliferation to replenish the β-cell mass lost during the progression of the disease. However, the adequate development of such therapies requires the knowledge of the molecular mechanisms controlling the expression of PAX4 as well as the downstream effectors that could account for PAX4 action.

The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice

LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.

Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice.

Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis.

Transient PAX8 Expression in Islets During Pregnancy Correlates With Beta Cell Survival Revealing a Novel Candidate Gene in Gestational Diabetes Mellitus

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8. A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM.