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We have previously shown that isolated islets embedded in type 1 collagen gel in the presence of a defined medium undergo transdifferentiation within 96 h to duct epithelial structures. The aim of this study was to identify the factors implicated in this process. Freshly isolated canine islets were embedded in type 1 collagen gel, Matrigel or agarose for up to 120 h and cultured in (i) Dulbecco's modified Eagle's medium (DMEM)/F12 plus cholera toxin (CT), (ii) medium CMRL1066 plus CT, (iii) CMRL1066 plus forskolin and (iv) CMRL1066 alone. At 16 h, intracellular levels of cAMP (fmol/10(3) islets) were increased in groups i-iii (642+/-17, 338+/-48, 1128+/-221) compared with group iv (106+/-19, P<0.01). Epithelial differentiation correlated with the total amount of intracellular cAMP measured over 120 h. Islet-epithelial transformation during the initial 36 h was associated with a wave of apoptosis which was followed by a wave of cell proliferation. During epithelial differentiation there was a progressive loss of all islet hormones and the concomitant expression of cytoskeletal proteins characteristic of duct epithelial cells. Islets in collagen and Matrigel demonstrated high rates of epithelial differentiation (63+/-2% and 71+/-4% respectively) compared with those in agarose gel (0+/-0%, P<0.001). Islets suspended in DMEM/F12 plus CT supplemented with soluble laminin or fibronectin did not undergo transformation. Prior incubation of freshly isolated islets with an integrin-binding arginine-glycine-aspartate motif-presenting synthetic peptide also reduced islet transformation. These studies confirm the biological potential of islets of Langerhans to differentiate to duct epithelial structures. cAMP-mediated signal transduction and an appropriate integrin-matrix interaction are necessary for this process to proceed.

The limitation of available islets for transplantation is a major obstacle for the treatment of diabetes through islet therapy. However, islet monolayers expanded ex vivo may provide a source of progenitor cells and a model to help understand islet development from precursor cell types. The existence of progenitor cells within the islets is highly likely, yet, to date, no fully defined or characterized postnatal stem cell has been isolated, expanded or marked. Our study evaluates the expression of progenitor markers, including the haematopoietic stem cell marker c-Kit, in epithelial monolayers derived from postnatal rat islets through immunofluorescence and RT-PCR, and the ability of precursor-rich monolayers to reform islet-like structures. Islets formed confluent monolayers when cultured on a type I collagen gel which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating c-Kit-expressing cells, with the proportion reaching a maximum of 45+/-6% at 8 weeks of culture. Evaluation of transcription factors at the mRNA level revealed constant PDX-1, ngn3 and Pax4 expression, while undifferentiated cell markers, such as Oct4 and alpha-fetoprotein, were also detected frequently after 4 weeks of culture. Changing the extracellular matrix protein to laminin-rich Matrigel, the monolayers re-formed islet-like clusters that secreted insulin in a glucose-responsive fashion. Our data show that islets can be expanded ex vivo to form epithelial monolayers with rich undifferentiating cell populations that are characterized by cells expressing the progenitor markers. These monolayers are capable of extensive proliferation and retain plasticity to form new islet cells, and c-Kit-expressing cells may play an important role in new islet cluster formation.

There has over the last several years been renewed interest in developing a system for generating new islets and a search for a self-renewing population in the pancreas. In particular, the neural stem cell marker nestin has been implicated as an islet precursor marker and its immunoreactivity has been localized in the islets of Langerhans. This study examines islet-derived epithelial monolayers expanded ex vivo to provide a source of nestin-expressing progenitor cells – a model that will help us understand the role of nestin-expressing cells in islet cell development. When cultured on a type I collagen gel, islets formed confluent monolayers which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating nestin-expressing cells, reaching a maximum of 54±10%. Co-expression studies demonstrated that the nestin-positive cells are heterogeneous, with some nestin-expressing cells co-localizing with the transcription factor PDX-1 and glucose transporter type 2 or lack of co-expression with vimentin. When clonal populations of nestin-positive cells were expanded and subjected to a differentiation protocol, only a population that expressed the transcription factor PDX-1 at the mRNA level was capable of re-expressing insulin at the mRNA and protein level. In conclusion, these studies demonstrate that expanded nestin-expressing cells in vitro from islet-derived epithelial monolayers are heterogeneous; clonal analysis of nestin-positive cells reveals that a distinct subpopulation of nestin/PDX-1-expressing cells is capable of forming insulin-producing cells.

Hepatocyte growth factor (HGF) has been suggested to be a potent regulator of β-cell function and proliferation. The purpose of this study was to investigate whether HGF could regulate the proliferation and differentiation of islet-derived epithelial monolayers into insulin-producing cells. We have generated islet-derived epithelial monolayers that are enriched with cells expressing c-Kit, a tyrosine kinase receptor and putative marker, from isolated postnatal rat islets. Monolayers were cultured on type I collagen gel and treated in defined differentiation medium with or without HGF (50 ng/ml) for 7 days. Subsequently, the expression of transcription factors and pancreatic endocrine cell markers as well as c-Kit expression were compared between the HGF (HGF⁺), no HGF treatment (HGF⁻) and monolayers without differentiation medium (control) groups, using immunocytochemical and RT-PCR approaches. We observed that the number of c-Kit-, glucose transport type 2 (Glut2)- and the transcription factor pancreatic duodenal homeobox-1 (PDX-1)-expressing cells were significantly increased in the HGF⁺ group. The expression of insulin at the mRNA and protein level was also increased in this treatment group with a 1.7-fold increase in basal insulin release and a 2.3-fold increase in insulin content in comparison with the HGF⁻ group. A high proliferative capacity was also found in the HGF⁺ group. Co-localization of insulin and PDX-1 or Glut2 was revealed frequently in cells treated with HGF⁺ with occasional co-staining of c-Kit and insulin observed. This study showed that HGF can activate the proliferation and differentiation of islet-derived epithelial monolayer into insulin-producing cells. However, no formation of islet-like clusters was observed. Taken together, this study implies that HGF mediates differentiation of immature cell types into insulin-expressing cells; however, HGF supplementation alone is insuffcient in restoring full β-cell function.

The development of fetal thyroid function is dependent on the embryogenesis, differentiation, and maturation of the thyroid gland. This is coupled with evolution of the hypothalamic–pituitary–thyroid axis and thyroid hormone metabolism, resulting in the regulation of thyroid hormone action, production, and secretion. Throughout gestation there is a steady supply of maternal thyroxine (T₄) which has been observed in embryonic circulation as early as 4 weeks post-implantation. This is essential for normal early fetal neurogenesis. Triiodothyronine concentrations remain very low during gestation due to metabolism via placental and fetal deiodinase type 3. T₄ concentrations are highly regulated to maintain low concentrations, essential for protecting the fetus and reaching key neurological sites such as the cerebral cortex at specific developmental stages. There are many known cell membrane thyroid hormone transporters in fetal brain that play an essential role in regulating thyroid hormone concentrations in key structures. They also provide the route for intracellular thyroid hormone interaction with associated thyroid hormone receptors, which activate their action. There is a growing body of experimental evidence from rats and humans to suggest that even mild maternal hypothyroxinemia may lead to abnormalities in fetal neurological development. Our review will focus on the ontogeny of thyroid hormone in fetal development, with a focus on cell membrane transporters and TR action in the brain.

Neurons controlling appetite are located in the hypothalamic arcuate nuclei (ARH). Offspring appetite regulation has been shown to be modified by dysregulation of ARH nuclear development. Most ARH developmental studies have been in altricial rodents whose hypothalamic development is predominantly postnatal. In primates including humans, much development of hypothalamic appetite regulatory centers occurs before birth. We hypothesized that i) appetitive peptides are abundantly expressed by 90 percent gestation (0.9G), ready for postnatal function; ii) by 0.9G, intrauterine growth restriction (IUGR) increases the orexigenic:anorexigenic peptide ratio; iii) IUGR increases fetal glucocorticoid receptor (GR) expression; and iv) IUGR decreases STAT3, which signals inhibition of appetite. We developed a fetal baboon IUGR model resulting from reduced maternal nutrition. Pregnant baboons were fed ad libitum, controls (CTR; n=24), or 70% CTR diet to produce IUGR (n=14). C-section was performed at 0.9G. In CTR (n=7) and IUGR (n=6) fetal brains, ARH appetite regulatory peptides (neuropeptide Y (NPY) and proopiomelanocortin (POMC)) were quantified immunohistochemically. Fetal plasma cortisol was raised in IUGR fetuses. We observed that NPY and POMC were well expressed by 0.9G. IUGR increased NPY, GR, and active phosphorylated GR and decreased POMC and phosphorylated form of STAT3. We conclude that IUGR dysregulates ARH development in ways that will reset the appetitive neuropeptide balance in favor of increased appetite drive in postnatal life. We postulate that changes in peptide abundance are in part due to increased fetal cortisol and ARH GR. These changes may contribute to predisposition to obesity in IUGR offspring.

Fat remodeling has been extensively explored through protein deacetylation, but not yet acetylation, as a viable therapeutic approach in the management of obesity and related metabolic disorders. Here, we investigated the functions of key acetyltransferases CBP/p300 in adipose remodeling and their physiological effects by generating adipose-specific deletion of CBP (Cbp-AKO), p300 (p300-AKO) and double-knockout (Cbp/p300-AKO) models. We demonstrated that Cbp-AKO exhibited marked brown remodeling of inguinal WAT (iWAT) but not epididymal WAT (eWAT) after cold exposure and that this pattern was exaggerated in diet-induced obesity (DIO). Despite this striking browning phenotype, loss of Cbp was insufficient to impact body weight or glucose tolerance. In contrast, ablation of p300 in adipose tissues had minimal effects on fat remodeling and adiposity. Surprisingly, double-knockout mice (Cbp/p300-AKO) developed severe lipodystrophy along with marked hepatic steatosis, hyperglycemia and hyperlipidemia. Furthermore, we demonstrated that pharmacological inhibition of Cbp and p300 activity suppressed adipogenesis. Collectively, these data suggest that (i) CBP, but not p300, has distinct functions in regulating fat remodeling and that this occurs in a depot-selective manner; (ii) brown remodeling occurs independently of the improvements in glucose metabolism and obesity and (iii) the combined roles of CBP and p300 are indispensable for normal adipose development.

p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKT^S473/T308 and S6K1^T389 phosphorylation but decreased S6^S235/236 and IRS-1^S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKT^S473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.