RFX6 regulates human intestinal patterning and function upstream of PDX1.

The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.

Nonsense mutation in the novel PERCC1 gene as a genetic cause of congenital diarrhea and enteropathy.

Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the 'intestine-critical region (ICR)', were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated two unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Program. Currently 12 and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents. In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C > G stop gain variant. We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That both parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.

Using Human Induced Pluripotent Stem Cell-Derived Organoids to Identify New Pathologies in Patients With PDX1 Mutations.

BACKGROUND & AIMS: Two patients with homozygous mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea, and poor weight gain, the causes of which were not identified through routine clinical testing. We aimed to perform a deep analysis of the stomach and intestine using organoids derived from induced pluripotent stem cells from PDX1188delC/188delC patients. METHODS: Gastric fundic, antral, and duodenal organoids were generated using induced pluripotent stem cell lines from a PDX1188delC/188delC patient and an isogenic induced pluripotent stem cell line where the PDX1 point mutation was corrected. RESULTS: Patient-derived PDX1188delC/188delC antral organoids exhibited an intestinal phenotype, whereas intestinal organoids underwent gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsy specimens from both PDX1188delC/188delC patients, which recapitulated the organoid phenotypes. Moreover, antral biopsy specimens also showed increased parietal cells and lacked G cells, suggesting loss of antral identity. All organoid pathologies were reversed upon CRISPR-mediated correction of the mutation. CONCLUSIONS: These patients will now be monitored for the progression of metaplasia and gastrointestinal complications that might be related to the reduced gastric and intestinal endocrine cells. This study demonstrates the utility of organoids in diagnosing uncovered pathologies.

Multifocal Insulinoma as the Unique Presenting Feature of Multiple Endocrine Neoplasia Type 1 in an Adolescent.

INTRODUCTION: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited disorder defined by the presence of two of the following endocrinopathies: primary hyperparathyroidism, anterior pituitary tumors, and duodenopancreatic neuroendocrine tumors (NETs). NETs, which can secrete hormones including insulin, gastrin, and glucagon, among others, are common in patients with MEN1 and are a major cause of morbidity and premature death. NETs are more common later in life, with very few cases described in children. Here, we describe a unique case of an adolescent with multifocal pancreatic NETs as the single presenting feature of MEN1. CASE PRESENTATION: A 13-year-old healthy male presented with severe weakness, altered mental status, and syncope in the setting of a venous blood glucose (BG) of 36 mg/dL. Workup showed an elevated insulin level (14 µIU/mL) when BG was 39 mg/dL with positive response to glucagon, concerning for hyperinsulinism. Diazoxide and chlorothiazide were started but not well tolerated secondary to emesis. Three suspected NETs were identified by magnetic resonance imaging and 68-Ga DOTATATE PET-CT imaging, including the largest, a 2.1 cm mass in the pancreatic head. A fourth mass in the pancreatic tail was identified via intraoperative ultrasound. All lesions were successfully enucleated and excised, and glucose levels normalized off diazoxide by post-op day 2. While the primary lesion stained for insulin and somatostatin by immunofluorescence (IF), consistent with his clinical presentation, the additional tumors expressed glucagon, somatostatin, pancreatic polypeptide, and chromogranin A but were negative for insulin. Genetic testing confirmed a pathogenic heterozygous mutation in MEN1 (c.969C>A, p.Tyr323). He had no other signs of MEN-associated comorbidities on screening. DISCUSSION/CONCLUSION: This case demonstrates that young patients with MEN1 can present with multifocal NETs. These NETs may have polyhormonal expression patterns despite a clinical presentation consistent with one primary hormone. Our patient had clinical symptoms and laboratory evaluation consistent with an insulinoma but was found to have four NETs, each with different IF staining patterns. Advanced preoperative and intraoperative imaging is important to identify and treat all present NETs. Moreover, serum hormone levels pre- and posttreatment could help evaluate whether NETs are actively secreting hormones into the bloodstream or simply expressing them within the pancreas. Finally, this case highlights the importance of genetic testing for MEN1 in all young patients with insulinomas.

Dual Role of Caspase 8 in Adipocyte Apoptosis and Metabolic Inflammation.

Caspases are cysteine-aspartic proteases that were initially discovered to play a role in apoptosis. However, caspase 8, in particular, also has additional nonapoptotic roles, such as in inflammation. Adipocyte cell death and inflammation are hypothesized to be initiating pathogenic factors in type 2 diabetes. Here, we examined the pleiotropic role of caspase 8 in adipocytes and obesity-associated insulin resistance. Caspase 8 expression was increased in adipocytes from mice and humans with obesity and insulin resistance. Treatment of 3T3-L1 adipocytes with caspase 8 inhibitor Z-IETD-FMK decreased both death receptor-mediated signaling and targets of nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling. We generated novel adipose tissue and adipocyte-specific caspase 8 knockout mice (aP2Casp8-/- and adipoqCasp8-/-). Both males and females had improved glucose tolerance in the setting of high-fat diet (HFD) feeding. Knockout mice also gained less weight on HFD, with decreased adiposity, adipocyte size, and hepatic steatosis. These mice had decreased adipose tissue inflammation and decreased activation of canonical and noncanonical NF-κB signaling. Furthermore, they demonstrated increased energy expenditure, core body temperature, and UCP1 expression. Adipocyte-specific activation of Ikbkb or housing mice at thermoneutrality attenuated improvements in glucose tolerance. These data demonstrate an important role for caspase 8 in mediating adipocyte cell death and inflammation to regulate glucose and energy homeostasis. ARTICLE HIGHLIGHTS: Caspase 8 is increased in adipocytes from mice and humans with obesity and insulin resistance. Knockdown of caspase 8 in adipocytes protects mice from glucose intolerance and weight gain on a high-fat diet. Knockdown of caspase 8 decreases Fas signaling, as well as canonical and noncanonical nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling in adipose tissue. Improved glucose tolerance occurs via reduced activation of NF-κB signaling and via induction of UCP1 in adipocytes.

Inhibition of Gsk3ß activity improves ß-cell function in c-KitWv/+ male mice.

Previous studies have shown that the stem cell marker, c-Kit, is involved in glucose homeostasis. We recently reported that c-Kit(Wv/+) male mice displayed the onset of diabetes at 8 weeks of age; however, the mechanisms by which c-Kit regulates ß-cell proliferation and function are unknown. The purpose of this study is to examine if c-Kit(Wv/+) mutation-induced ß-cell dysfunction is associated with downregulation of the phospho-Akt/Gsk3ß pathway in c-Kit(Wv/+) male mice. Histology and cell signaling were examined in C57BL/6J/Kit(Wv/+) (c-Kit(Wv/+)) and wild-type (c-Kit(+/+)) mice using immunofluorescence and western blotting approaches. The Gsk3ß inhibitor, 1-azakenpaullone (1-AKP), was administered to c-Kit(Wv/+) and c-Kit(+/+) mice for 2 weeks, whereby alterations in glucose metabolism were examined and morphometric analyses were performed. A significant reduction in phosphorylated Akt was observed in the islets of c-Kit(Wv/+) mice (P<0.05) along with a decrease in phosphorylated Gsk3ß (P<0.05), and cyclin D1 protein level (P<0.01) when compared with c-Kit(+/+) mice. However, c-Kit(Wv/+) mice that received 1-AKP treatment demonstrated normal fasting blood glucose with significantly improved glucose tolerance. 1-AKP-treated c-Kit(Wv/+) mice also showed increased ß-catenin, cyclin D1 and Pdx-1 levels in islets, demonstrating that inhibition of Gsk3ß activity led to increased ß-cell proliferation and insulin secretion. These data suggest that c-Kit(Wv/+) male mice had alterations in the Akt/Gsk3ß signaling pathway, which lead to ß-cell dysfunction by decreasing Pdx-1 and cyclin D1 levels. Inhibition of Gsk3ß could prevent the onset of diabetes by improving glucose tolerance and ß-cell function.

Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.

Human organoid model systems lack important cell types that, in the embryo, are incorporated into organ tissues during development. We developed an organoid assembly approach starting with cells from the three primary germ layers-enteric neuroglial, mesenchymal, and epithelial precursors-that were derived separately from human pluripotent stem cells (PSCs). From these three cell types, we generated human antral and fundic gastric tissue containing differentiated glands surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered antral tissue. Using this experimental system, we show that human enteric neural crest cells (ENCCs) promote mesenchyme development and glandular morphogenesis of antral stomach organoids. Moreover, ENCCs can act directly on the foregut to promote a posterior fate, resulting in organoids with a Brunner's gland phenotype. Thus, germ layer components that are derived separately from PSCs can be used for tissue engineering to generate complex human organoids.

Adiponectin-induced ERK and Akt phosphorylation protects against pancreatic beta cell apoptosis and increases insulin gene expression and secretion.

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin-induced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 µg/ml) or full-length (5 µg/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1- and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.

c-Kit in early onset of diabetes: a morphological and functional analysis of pancreatic beta-cells in c-KitW-v mutant mice.

c-Kit tyrosine receptor kinase, a well-established stem cell marker, is expressed in a variety of tissues including the pancreas. The involvement of c-Kit in fetal rat and human endocrine pancreatic development, survival, and function has been well characterized but primarily using in vitro experimental approaches. Therefore, the aim of the current study was to examine whether deficiency of a functional c-Kit receptor would have physiological and functional implications in vivo. We characterized the c-Kit mutant mouse, c-Kit(W-v/+), to evaluate the in vivo role of c-Kit in beta-cell growth and function. Here we report that male c-Kit(W-v/+) mice, at 8 wk of age, showed high fasting blood glucose levels and impaired glucose tolerance, which was associated with low levels of insulin secretion after glucose stimulation in vivo and in isolated islets. Morphometric analysis revealed that beta-cell mass was significantly reduced (50%) in male c-Kit(W-v/+) mice when compared with controls (c-Kit(+/+)) (P < 0.05). In parallel, a reduction in pancreatic duodenal homeobox-1 and insulin gene expression in whole pancreas as well as isolated islets of c-Kit(W-v/+) male mice was noted along with a decrease in pancreatic insulin content. Furthermore, the reduction in beta-cell mass in male c-Kit(W-v/+) mice was associated with a decrease in beta-cell proliferation. Interestingly, these changes were not observed in female c-Kit(W-v/+) mice until 40 wk of age. Our results clearly demonstrate that the c-Kit receptor is involved in the regulation of glucose metabolism, likely through an important role in beta-cell development and function.

FAK signalling controls insulin sensitivity through regulation of adipocyte survival.

Focal adhesion kinase (FAK) plays a central role in integrin signalling, which regulates growth and survival of tumours. Here we show that FAK protein levels are increased in adipose tissue of insulin-resistant obese mice and humans. Disruption of adipocyte FAK in mice or in 3T3 L1 cells decreases adipocyte survival. Adipocyte-specific FAK knockout mice display impaired adipose tissue expansion and insulin resistance on prolonged metabolic stress from a high-fat diet or when crossed on an obese db/db or ob/ob genetic background. Treatment of these mice with a PPARγ agonist does not restore adiposity or improve insulin sensitivity. In contrast, inhibition of apoptosis, either genetically or pharmacologically, attenuates adipocyte death, restores normal adiposity and improves insulin sensitivity. Together, these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the context of adipose tissue expansion as a result of caloric excess.

A novel insulin receptor mutation in an adolescent with acanthosis nigricans and hyperandrogenism.

Insulin receptor mutations cause extreme insulin resistance resulting in acanthosis nigricans and hyperandrogenism. We report a pre-menarchal adolescent female with normal weight, with severe acanthosis nigricans, acne, and hirsutism. Initial investigation revealed elevated fasting and post-prandial insulin and high testosterone and androstenedione levels. Her father had frequent complaints of hypoglycemia. Coding sequence and splice junction analysis of the INSR gene, in our patient and her father, revealed a heterozygous missense mutation in the ß subunit of the insulin receptor (Arg1131Trp), resulting in receptor loss of function. Metformin therapy and carbohydrate control improved acanthosis and menarche ensued within 3 months. Our case highlights the importance of distinguishing insulin resistance commonly associated with obesity from monogenic defects. Although, there is no consensus on treatment of children with monogenic forms of insulin resistance due to its rarity, dietary and lifestyle modifications and insulin-sensitizing agents play a key role in management.

The role of SOX9 transcription factor in pancreatic and duodenal development.

Progenitor expansion during development is a highly regulated process dictating the final organ size, while expansion of specific progenitor populators can adjust the final cellular composition of the organ. Understanding factors involved in these pathways is required to develop cell-based therapies such as ß-cell transplantation for conditions such as diabetes mellitus. One versatile factor controlling both processes as well as a network of other proteins involved in pancreatic and duodenal development is the transcription factor SOX9. This review will focus on a comparison of SOX9 function during progenitor expansion and differentiation in the developing pancreas and duodenum with specific focus on endocrine development. During human pancreatic development, SOX9 functions in a dose-dependent manner to regulate epithelial progenitor expansion and endocrine differentiation. SOX9 expression is eventually limited to a subset of ductal and centroacinar cells, hypothesized to be the pancreatic stem cell compartment. Similarly, during duodenal development, SOX9 is expressed in most early epithelial progenitors and becomes gradually restricted to proliferative progenitors in the lower crypts, as well as mature Paneth and enteroendocrine cells indicating some differences in functional roles. However, in both developmental contexts, SOX9 is involved in pathways responsible for cellular proliferation and differentiation, such as Notch and Wnt. With its adaptable and central function in progenitor control, SOX9 represents an attractive target for manipulation for in vitro progenitor expansion and differentiation meriting further investigation.

SOX9 regulates endocrine cell differentiation during human fetal pancreas development.

The transition of pancreatic progenitor cells to mature endocrine cells is regulated by the sequential activation and interaction of several transcription factors. In mice, the transcription factor Sox9 has been shown to support endocrine cell differentiation. However, the functional role of SOX9 during pancreas development in the human has yet to be determined. The present study was to characterize SOX9 expression during human fetal pancreas development and examine its functional role by transfection with SOX9 siRNA or SOX9 expression vectors. Here we report that SOX9 was most frequently expressed in PDX1(+) cells (60-83%) and least in mature endocrine cells (<1-14%). The proliferation of SOX9(+) cells was significantly higher at 8-10 weeks than at 14-21 weeks (p<0.05) or 20-21 weeks (p<0.01). SOX9 frequently co-localized with FOXA2, NGN3 and transcription factors linked to NGN3 (NKX2.2, NKX6.1, PAX6). siRNA knockdown of SOX9 significantly decreased islet-epithelial cell proliferation, NGN3, NKX6.1, PAX6 and INS mRNA levels and the number of NGN3(+) and insulin(+) cells (p<0.05) while increasing GCG mRNA and glucagon(+) cells (p<0.05). Examination of SOX9 associated signaling pathways revealed a decrease in phospho-Akt (p<0.01), phospho-GSK3ß (p<0.01) and cyclin D1 (p<0.01) with a decrease in nuclear ß-catenin(+) (p<0.05) cells following SOX9 siRNA knockdown. In contrast, over-expression of SOX9 significantly increased the number of islet cells proliferating, NGN3, NKX6.1, PAX6 and INS mRNA levels, the phospho-Akt/GSK3ß cascade and the number of insulin(+) cells. Our results demonstrated that SOX9 is important for the expression of NGN3 and molecular markers of endocrine cell differentiation in the human fetal pancreas.

Integrin {alpha}3, but not {beta}1, regulates islet cell survival and function via PI3K/Akt signaling pathways.

ß1-integrin is a well-established regulator of ß-cell activities; however, the role of its associated α-subunits is relatively unknown. Previously, we have shown that human fetal islet and INS-1 cells highly express α3ß1-integrin and that collagens I and IV significantly enhance their survival and function; in addition, blocking ß1 function in the fetal islet cells decreased adhesion on collagen I and increased apoptosis. The present study investigates the effect of blocking α3. Using α3 blocking antibody or small interfering RNA, the effects of α3-integrin blockade were examined in isolated human fetal or adult islet cells or INS-1 cells, cultured on collagens I or IV. In parallel, ß1 blockade was analyzed in INS-1 cells. Perturbing α3 function in human islet or INS-1 cells resulted in significant decreases in cell function (adhesion, spreading, proliferation and Pdx1 and insulin expression/secretion), primarily on collagen IV. A significant decrease in focal adhesion kinase and ERK1/2 phosphorylation and increased caspase3 cleavage were observed on both collagens. These effects were similar to changes after ß1 blockade. Interestingly, only α3 blockade reduced expression of phospho-Akt and members of its downstream signaling cascades (glycogen synthase kinase ß and X-linked inhibitor of apoptosis), demonstrating a specific effect of α3 on the phosphatidylinositol 3-kinase/Akt pathway. These results suggest that α3- as well as ß1-integrin-extracellular matrix interactions are critical for modulating ß-cell survival and function through specialized signaling cascades and enhance our understanding of how to improve islet microenvironments for cell-based treatments of diabetes.

Integrins and extracellular matrices in pancreatic tissue engineering.

The role of integrin receptors in regulating numerous cellular programs have been well studied in the endocrine pancreas. These adhesion receptors and their interactions with the extracellular matrix (ECM) are important determinants of islet cell biology as they influence the development, survival and function of the islets of Langerhans. In this review, we will discuss the profound role of integrin-ECM relationships in controlling pancreatic tissue morphogenesis and the anti-apoptotic properties that these receptors confer through their dynamic and unique signaling pathways. Relationships between the ECM-integrin receptors will also be discussed in light of islet-based therapies including transplantation procedures and pancreatic tissue engineering initiatives.

The emerging role of SOX transcription factors in pancreatic endocrine cell development and function.

The transition of pancreatic progenitor cells to mature beta cells is regulated by the interaction of several transcription factors, including members of the sex-determining region on Y box (SOX) family of transcription factors. The SOX proteins are widely involved in cell fate determination and the development of several tissues, including bone, heart, gonads, lymphocytes, and glial cells as well as the pancreas. In this review, we will present recent findings that illustrate the critical role of SOX transcription factors in maintaining pancreatic progenitor cell pools and in controlling pancreatic islet morphogenesis and islet function. Interrelationships between the SOX family and other pancreatic transcription factors specific to endocrine lineages will also be discussed in light of islet cell-based therapies for the treatment of diabetes.

Expression and function of alphabeta1 integrins in pancretic beta (INS-1) cells.

Integrin-extracellular matrix interactions are important determinants of beta cell behaviours. The beta1 integrin is a well-known regulator of beta cell activities; however, little is known of its associated alpha subunits. In the present study, alphabeta1 integrin expression was examined in the rat insulinoma cell line (INS-1) to identify their role in beta cell survival and function. Seven alpha subunits associated with beta1 integrin were identified, including alpha1-6 and alphaV. Among these heterodimers, alpha3beta1 was most highly expressed. Common ligands for the alpha3beta1 integrin, including fibronectin, laminin, collagen I and collagen IV were tested to identify the most suitable matrix for INS-1 cell proliferation and function. Cells exposed to collagen I and IV demonstrated significant increases in adhesion, spreading, cell viability, proliferation, and FAK phosphorylation when compared to cells cultured on fibronectin, laminin and controls. Integrin-dependent attachment also had a beneficial effect on beta cell function, increasing Pdx-1 and insulin gene and protein expression on collagens I and IV, in parallel with increased basal insulin release and enhanced insulin secretion upon high glucose challenge. Furthermore, functional blockade of alpha3beta1 integrin decreased cell adhesion, spreading and viability on both collagens and reduced Pdx-1 and insulin expression, indicating that its interactions with collagen matrices are important for beta cell survival and function. These results demonstrate that specific alphabeta1 integrin-ECM interactions are critical regulators of INS-1 beta cell survival and function and will be important in designing optimal conditions for cell-based therapies for diabetes treatment.