Toll-like receptors TLR2 and TLR4 block the replication of pancreatic ß cells in diet-induced obesity.

Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.

Harnessing prostaglandin E2 signaling to ameliorate autoimmunity.

The etiology of most autoimmune diseases remains unknown; however, shared among them is a disruption of immunoregulation. Prostaglandin lipid signaling molecules possess context-dependent immunoregulatory properties, making their role in autoimmunity difficult to decipher. For example, prostaglandin E2 (PGE2) can function as an immunosuppressive molecule as well as a proinflammatory mediator in different circumstances, contributing to the expansion and activation of T cell subsets associated with autoimmunity. Recently, PGE2 was shown to play important roles in the resolution and post-resolution phases of inflammation, promoting return to tissue homeostasis. We propose that PGE2 plays both proinflammatory and pro-resolutory roles in the etiology of autoimmunity, and that harnessing this signaling pathway during the resolution phase might help prevent autoimmune attack.

The prostaglandin E2 EP3 receptor has disparate effects on islet insulin secretion and content in ß-cells in a high-fat diet-induced mouse model of obesity.

Signaling through prostaglandin E2 EP3 receptor (EP3) actively contributes to the ß-cell dysfunction of type 2 diabetes (T2D). In T2D models, full-body EP3 knockout mice have a significantly worse metabolic phenotype than wild-type controls due to hyperphagia and severe insulin resistance resulting from loss of EP3 in extra-pancreatic tissues, masking any potential beneficial effects of EP3 loss in the ß cell. We hypothesized ß-cell-specific EP3 knockout (EP3 ßKO) mice would be protected from high-fat diet (HFD)-induced glucose intolerance, phenocopying mice lacking the EP3 effector, Gαz, which is much more limited in its tissue distribution. When fed a HFD for 16 wk, though, EP3 ßKO mice were partially, but not fully, protected from glucose intolerance. In addition, exendin-4, an analog of the incretin hormone, glucagon-like peptide 1, more strongly potentiated glucose-stimulated insulin secretion in islets from both control diet- and HFD-fed EP3 ßKO mice as compared with wild-type controls, with no effect of ß-cell-specific EP3 loss on islet insulin content or markers of replication and survival. However, after 26 wk of diet feeding, islets from both control diet- and HFD-fed EP3 ßKO mice secreted significantly less insulin as a percent of content in response to stimulatory glucose, with or without exendin-4, with elevated total insulin content unrelated to markers of ß-cell replication and survival, revealing severe ß-cell dysfunction. Our results suggest that EP3 serves a critical role in temporally regulating ß-cell function along the progression to T2D and that there exist Gαz-independent mechanisms behind its effects.NEW & NOTEWORTHY The EP3 receptor is a strong inhibitor of ß-cell function and replication, suggesting it as a potential therapeutic target for the disease. Yet, EP3 has protective roles in extrapancreatic tissues. To address this, we designed ß-cell-specific EP3 knockout mice and subjected them to high-fat diet feeding to induce glucose intolerance. The negative metabolic phenotype of full-body knockout mice was ablated, and EP3 loss improved glucose tolerance, with converse effects on islet insulin secretion and content.

Initiation of Metformin in Early Pregnancy Results in Fetal Bioaccumulation, Growth Restriction & Renal Dysmorphology in a Primate Model.

BACKGROUND: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes, type 2 diabetes, gestational diabetes and (most recently) pre-eclampsia. With its expanded use, however, concerns of unintended harm have been raised. OBJECTIVE: We developed an experimental primate model and applied triple-quadruple pole LC mass spectrometry (UHPLC-QQQ) for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. STUDY DESIGN: Within 30 days of confirmed conception (defined as early pregnancy), n=13 time-bred (TMB) Rhesus dams with gestations designated for fetal necropsy were initiated on twice daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet (WSD). Metformin or placebo vehicle control were delivered in a variety of treats while animals were separated via a slide. A Cesarean was performed at G145, and amniotic fluid and blood were collected and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated UHPLC-QQQ in SRM against standard curves. RESULTS: Among the n=13 G145 pregnancies with fetal necropsy, n=1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 µM metformin/kg maternal weight or fetal/placental tissue), while a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight 248.32 g, <1%) and was suspected of having a fetal congenital condition. After excluding these two fetal gestations from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4, 3 female, 1 male fetuses) or 10 mg/kg metformin (n=7, 5 female, 2 male fetuses) were available for analyses. Among dams initiated on metformin by G30 (regardless of maternal diet), we observed significant bioaccumulation within the fetal kidney (0.78-6.06 µmol/kg, mean 2.48 µmol/kg) , liver (0.16-0.73 µmol/kg, mean 0.38 µmol/kg), fetal gut (0.28-1.22 µmol/kg, mean 0.70 µmol/kg), amniotic fluid (0.43-3.33 µmol/L, mean 1.88 µmol/L), placenta (0.16-1.0 µmol/kg , mean 0.50 µmol/kg) and fetal serum (0 -0.66 µmol/L , mean 0.23 µmol/L ), and fetal urine (4.1-174.1 µmol/L mean 38.5 µmol/L ), with fetal levels near biomolar equivalent to maternal levels (maternal serum 0.18-0.86 µmol/L , mean 0.46 µmol/L; maternal urine 42.6-254.0 µmol/L , mean 149.3 µmol/L). WSD feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta nor fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (p<0.05), collectively driving lower delivery weight (p<0.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness; this renal dysmorphology was not accompanied by structural nor functional changes indicative of renal insufficiency. CONCLUSIONS: We demonstrate fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology following maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

HNF6 and Rev-erbα integrate hepatic lipid metabolism by overlapping and distinct transcriptional mechanisms.

Hepatocyte nuclear factor 6 (HNF6) is required for liver development, but its role in adult liver metabolism is not known. Here we show that deletion of HNF6 in livers of adult C57Bl/6 mice leads to hepatic steatosis in mice fed normal laboratory chow. Although HNF6 is known mainly as a transcriptional activator, hepatic loss of HNF6 up-regulated many lipogenic genes bound directly by HNF6. Many of these genes are targets of the circadian nuclear receptor Rev-erbα, and binding of Rev-erbα at these sites was lost when HNF6 was ablated in the liver. While HNF6 and Rev-erbα coordinately regulate hepatic lipid metabolism, each factor also affects additional gene sets independently. These findings highlight a novel mechanism of transcriptional repression by HNF6 and demonstrate how overlapping and distinct mechanisms of transcription factor function contribute to the integrated physiology of the liver.

Context-dependent effects of CCN2 on ß-cell mass expansion and indicators of cell stress in the setting of acute and chronic stress.

Stimulation of functional ß-cell mass expansion can be beneficial for the treatment of type 2 diabetes. Our group has previously demonstrated that the matricellular protein CCN2 can induce ß-cell mass expansion during embryogenesis, and postnatally during pregnancy and after 50% ß-cell injury. The mechanism by which CCN2 stimulates ß-cell mass expansion is unknown. However, CCN2 does not induce ß-cell proliferation in the setting of euglycemic and optimal functional ß-cell mass. We thus hypothesized that ß-cell stress is required for responsiveness to CCN2 treatment. In this study, a doxycycline-inducible ß-cell-specific CCN2 transgenic mouse model was utilized to evaluate the effects of CCN2 on ß-cell stress in the setting of acute (thapsigargin treatment ex vivo) or chronic [high-fat diet or leptin receptor haploinsufficiency (db/+) in vivo] cellular stress. CCN2 induction during 1 wk or 10 wk of high-fat diet or in db/+ mice had no effect on markers of ß-cell stress. However, CCN2 induction did result in a significant increase in ß-cell mass over high-fat diet alone when animals were fed high-fat diet for 10 wk, a duration known to induce insulin resistance. CCN2 induction in isolated islets treated with thapsigargin ex vivo resulted in upregulation of the gene encoding the Nrf2 transcription factor, a master regulator of antioxidant genes, suggesting that CCN2 further activates this pathway in the presence of cell stress. These studies indicate that the potential of CCN2 to induce ß-cell mass expansion is context-dependent and that the presence of ß-cell stress does not ensure ß-cell proliferation in response to CCN2.NEW & NOTEWORTHY CCN2 promotes ß-cell mass expansion in settings of suboptimal ß-cell mass. Here, we demonstrate that the ability of CCN2 to induce ß-cell mass expansion in the setting of ß-cell stress is context-dependent. Our results suggest that ß-cell stress is necessary but insufficient for CCN2 to increase ß-cell proliferation and mass. Furthermore, we found that CCN2 promotes upregulation of a key antioxidant transcription factor, suggesting that modulation of ß-cell oxidative stress contributes to the actions of CCN2.

Maternal Western-style diet in nonhuman primates leads to offspring islet adaptations including altered gene expression and insulin hypersecretion.

Maternal overnutrition is associated with increased susceptibility to type 2 diabetes in the offspring. Rodent models have shown that maternal overnutrition influences islet function in offspring. To determine whether maternal Western-style diet (WSD) alters prejuvenile islet function in a model that approximates that of human offspring, we utilized a well-characterized Japanese macaque model. We compared islet function from offspring exposed to WSD throughout pregnancy and lactation and weaned to WSD (WSD/WSD) compared with islets from offspring exposed only to postweaning WSD (CD/WSD) at 1 yr of age. WSD/WSD offspring islets showed increased basal insulin secretion and an exaggerated increase in glucose-stimulated insulin secretion, as assessed by dynamic ex vivo perifusion assays, relative to CD/WSD-exposed offspring. We probed potential mechanisms underlying insulin hypersecretion using transmission electron microscopy to evaluate ß-cell ultrastructure, qRT-PCR to quantify candidate gene expression, and Seahorse assay to assess mitochondrial function. Insulin granule density, mitochondrial density, and mitochondrial DNA ratio were similar between groups. However, islets from WSD/WSD male and female offspring had increased expression of transcripts known to facilitate stimulus-secretion coupling and changes in the expression of cell stress genes. Seahorse assay revealed increased spare respiratory capacity in islets from WSD/WSD male offspring. Overall, these results show that maternal WSD feeding confers changes to genes governing insulin secretory coupling and results in insulin hypersecretion as early as the postweaning period. The results suggest a maternal diet leads to early adaptation and developmental programming in offspring islet genes that may underlie future ß-cell dysfunction.NEW & NOTEWORTHY Programed adaptations in islets in response to maternal WSD exposure may alter ß-cell response to metabolic stress in offspring. We show that islets from maternal WSD-exposed offspring hypersecrete insulin, possibly due to increased components of stimulus-secretion coupling. These findings suggest that islet hyperfunction is programed by maternal diet, and changes can be detected as early as the postweaning period in nonhuman primate offspring.

Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease.

Maturity-onset diabetes of the young type 5 (MODY5) is due to heterozygous mutations or deletion of HNF1B. No mouse models are currently available to recapitulate the human MODY5 disease. Here, we investigate the pancreatic phenotype of a unique MODY5 mouse model generated by heterozygous insertion of a human HNF1B splicing mutation at the intron-2 splice donor site in the mouse genome. This Hnf1bsp2/+ model generated with targeted mutation of Hnf1b mimicking the c.544+1G>T (T) mutation identified in humans, results in alternative transcripts and a 38% decrease of native Hnf1b transcript levels. As a clinical feature of MODY5 patients, the hypomorphic mouse model Hnf1bsp2/+ displays glucose intolerance. Whereas Hnf1bsp2/+ isolated islets showed no altered insulin secretion, we found a 65% decrease in pancreatic insulin content associated with a 30% decrease in total large islet volume and a 20% decrease in total ß-cell volume. These defects were associated with a 30% decrease in expression of the pro-endocrine gene Neurog3 that we previously identified as a direct target of Hnf1b, showing a developmental etiology. As another clinical feature of MODY5 patients, the Hnf1bsp2/+ pancreases display exocrine dysfunction with hypoplasia. We observed chronic pancreatitis with loss of acinar cells, acinar-to-ductal metaplasia, and lipomatosis, with upregulation of signaling pathways and impaired acinar cell regeneration. This was associated with ductal cell deficiency characterized by shortened primary cilia. Importantly, the Hnf1bsp2/+ mouse model reproduces the pancreatic features of the human MODY5/HNF1B disease, providing a unique in vivo tool for molecular studies of the endocrine and exocrine defects and to advance basic and translational research. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The Beta Cell in Type 2 Diabetes.

PURPOSE OF REVIEW: This review summarizes the alterations in the ß-cell observed in type 2 diabetes (T2D), focusing on changes in ß-cell identity and mass and changes associated with metabolism and intracellular signaling. RECENT FINDINGS: In the setting of T2D, ß-cells undergo changes in gene expression, reverting to a more immature state and in some cases transdifferentiating into other islet cell types. Alleviation of metabolic stress, ER stress, and maladaptive prostaglandin signaling could improve ß-cell function and survival. The ß-cell defects leading to T2D likely differ in different individuals and include variations in ß-cell mass, development, ß-cell expansion, responses to ER and oxidative stress, insulin production and secretion, and intracellular signaling pathways. The recent recognition that some ß-cells undergo dedifferentiation without dying in T2D suggests strategies to revive these cells and rejuvenate their functionality.

Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability.

The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in ß-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the ß-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal ß-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal ß-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous ß-cells would have an impaired ability to respond to stress. Indeed, we observed that ß-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.

Increases in bioactive lipids accompany early metabolic changes associated with ß-cell expansion in response to short-term high-fat diet.

Pancreatic ß-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult ß cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse ß-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD ß-cell expansion mouse model to identify pathways and metabolite changes associated with ß-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; ß-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced ß-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B5) decreased >2-fold. The results suggest that HFD-mediated ß-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.

Preserving Mafa expression in diabetic islet ß-cells improves glycemic control in vivo.

The murine Mafa transcription factor is a key regulator of postnatal islet ß-cell activity, affecting insulin transcription, insulin secretion, and ß-cell mass. Human MAFA expression is also markedly decreased in islet ß-cells of type 2 diabetes mellitus (T2DM) patients. Moreover, levels are profoundly reduced in db/db islet ß-cells, a mouse model of T2DM. To examine the significance of this key islet ß-cell-enriched protein to glycemic control under diabetic conditions, we generated transgenic mice that conditionally and specifically produced Mafa in db/db islet ß-cells. Sustained expression of Mafa resulted in significantly lower plasma glucose levels, higher plasma insulin, and augmented islet ß-cell mass. In addition, there was increased expression of insulin, Slc2a2, and newly identified Mafa-regulated genes involved in reducing ß-cell stress, like Gsta1 and Gckr. Importantly, the levels of human GSTA1 were also compromised in T2DM islets. Collectively, these results illustrate how consequential the reduction in Mafa activity is to islet ß-cell function under pathophysiological conditions.

Connective tissue growth factor is critical for proper ß-cell function and pregnancy-induced ß-cell hyperplasia in adult mice.

During pregnancy, maternal ß-cells undergo compensatory changes, including increased ß-cell mass and enhanced glucose-stimulated insulin secretion. Failure of these adaptations to occur results in gestational diabetes mellitus. The secreted protein connective tissue growth factor (CTGF) is critical for normal ß-cell development and promotes regeneration after partial ß-cell ablation. During embryogenesis, CTGF is expressed in pancreatic ducts, vasculature, and ß-cells. In adult pancreas, CTGF is expressed only in the vasculature. Here we show that pregnant mice with global Ctgf haploinsufficiency (Ctgf(LacZ/+)) have an impairment in maternal ß-cell proliferation; no difference was observed in virgin Ctgf(LacZ/+) females. Using a conditional CTGF allele, we found that mice with a specific inactivation of CTGF in endocrine cells (Ctgf(ΔEndo)) develop gestational diabetes during pregnancy, but this is due to a reduction in glucose-stimulated insulin secretion rather than impaired maternal ß-cell proliferation. Moreover, virgin Ctgf(ΔEndo) females also display impaired GSIS with glucose intolerance, indicating that underlying ß-cell dysfunction precedes the development of gestational diabetes in this animal model. This is the first time a role for CTGF in ß-cell function has been reported.

Sustained expression of GLP-1 receptor differentially modulates ß-cell functions in diabetic and nondiabetic mice.

Glucagon-like peptide 1 (GLP-1) has been shown to play important roles in maintaining ß-cell functions, such as insulin secretion and proliferation. While expression levels of GLP-1 receptor (Glp1r) are compromised in the islets of diabetic rodents, it remains unclear when and to what degree Glp1r mRNA levels are decreased during the progression of diabetes. In this study, we performed real-time PCR with the islets of db/db diabetic mice at different ages, and found that the expression levels of Glp1r were comparable to those of the islets of nondiabetic db/misty controls at the age of four weeks, and were significantly decreased at the age of eight and 12 weeks. To investigate whether restored expression of Glp1r affects the diabetic phenotypes, we generated the transgenic mouse model Pdx1(PB)-CreER(TM); CAG-CAT-Glp1r (ßGlp1r) that allows for induction of Glp1r expression specifically in ß cells. Whereas the expression of exogenous Glp1r had no measurable effect on glucose tolerance in nondiabetic ßGlp1r;db/misty mice, ßGlp1r;db/db mice exhibited higher glucose and lower insulin levels in blood on glucose challenge test than control db/db littermates. In contrast, four weeks of treatment with exendin-4 improved the glucose profiles and increased serum insulin levels in ßGlp1r;db/db mice, to significantly higher levels than those in control db/db mice. These differential effects of exogenous Glp1r in nondiabetic and diabetic mice suggest that downregulation of Glp1r might be required to slow the progression of ß-cell failure under diabetic conditions.

High-fat diet-induced ß-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.

Both short- (1 wk) and long-term (2-12 mo) high-fat diet (HFD) studies reveal enhanced ß-cell mass due to increased ß-cell proliferation. ß-Cell proliferation following HFD has been postulated to occur in response to insulin resistance; however, whether HFD can induce ß-cell proliferation independent of insulin resistance has been controversial. To examine the kinetics of HFD-induced ß-cell proliferation and its correlation with insulin resistance, we placed 8-wk-old male C57Bl/6J mice on HFD for different lengths of time and assayed the following: glucose tolerance, insulin secretion in response to glucose, insulin tolerance, ß-cell mass, and ß-cell proliferation. We found that ß-cell proliferation was significantly increased after only 3 days of HFD feeding, weeks before an increase in ß-cell mass or peripheral insulin resistance was detected. These results were confirmed by hyperinsulinemic euglycemic clamps and measurements of α-hydroxybutyrate, a plasma biomarker of insulin resistance in humans. An increase in expression of key islet-proliferative genes was found in isolated islets from 1-wk HFD-fed mice compared with chow diet (CD)-fed mice. These data indicate that short-term HFD feeding enhances ß-cell proliferation before insulin resistance becomes apparent.

Onecut1 is essential for horizontal cell genesis and retinal integrity.

Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (∼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.

Connective tissue growth factor (CTGF/CCN2) negatively regulates BMP-2 induced osteoblast differentiation and signaling.

Connective tissue growth factor (CTGF/CCN2) and bone morphogenetic protein (BMP)-2 are both produced and secreted by osteoblasts. Both proteins have been shown to have independent effects in regulating osteoblast proliferation, maturation and mineralization. However, how these two proteins interact during osteoblast differentiation remains unknown. In this study, we utilized two cell culture model systems, osteoblasts derived from CTGF knockout (KO) mice and osteoblasts infected with an adenovirus which over-expresses CTGF (Ad-CTGF), to investigate the effects of CTGF and BMP-2 on osteoblast development and function in vitro. Contrary to a previously published report, osteoblast maturation and mineralization were similar in osteogenic cultures derived from KO and WT calvaria in the absence of BMP-2 stimulation. Interestingly, in KO and WT osteoblast cultures stimulated with BMP-2, the KO osteoblasts exhibited enhanced osteoblast differentiation. This increase in osteoblast differentiation was accompanied by increased protein levels of phosphorylated Smad 1/5/8 and mRNA expression levels of bone morphogenetic protein receptor Ib. We also examined osteoblast differentiation in cultures that were infected with an adenoviral-CTGF vector (Ad-CTGF) and in controls. Continuous over-expression of CTGF resulted in decreased osteoblast maturation and mineralization in both unstimulated and BMP-2 stimulated cultures. Impaired osteoblast differentiation in cultures over-expressing CTGF was accompanied by decreased protein levels of phosphorylated Smad 1/5/8. Collectively, the data from these studies demonstrate that CTGF acts to negatively regulate BMP-2 induced signaling and osteoblast differentiation, and warrant additional studies to determine the precise mechanism(s) responsible for this effect. J. Cell. Physiol. 229: 672-681, 2014. © 2013 Wiley Periodicals, Inc.

Loss of HNF6 expression correlates with human pancreatic cancer progression.

Normal pancreatic epithelium progresses through various stages of pancreatic intraepithelial neoplasms (PanINs) in the development of pancreatic ductal adenocarcinoma (PDAC). Transcriptional regulation of this progression is poorly understood. In mouse, the hepatic nuclear factor 6 (Hnf6) transcription factor is expressed in ductal cells and at lower levels in acinar cells of the adult pancreas, but not in mature endocrine cells. Hnf6 is critical for terminal differentiation of the ductal epithelium during embryonic development and for pancreatic endocrine cell specification. We previously showed that, in mice, loss of Hnf6 from the pancreatic epithelium during organogenesis results in increased duct proliferation and altered duct architecture, increased periductal fibrosis and acinar-to-ductal metaplasia. Here we show that decreased expression of HNF6 is strongly correlated with increased severity of PanIN lesions in samples of human pancreata and is absent from >90% of PDAC. Mouse models in which cancer progression can be analyzed from the earliest stages that are seldom accessible in humans support a role for Hnf6 loss in progression from early- to late-stage PanIN and PDAC. In addition, gene expression analyses of human pancreatic cancer reveal decreased expression of HNF6 and its direct and indirect target genes compared with normal tissue and upregulation of genes that act in opposition to HNF6 and its targets. The negative correlation between HNF6 expression and pancreatic cancer progression suggests that HNF6 maintains pancreatic epithelial homeostasis in humans, and that its loss contributes to the progression from PanIN to ductal adenocarcinoma. Insight on the role of HNF6 in pancreatic cancer development could lead to its use as a biomarker for early detection and prognosis.

Connective tissue growth factor acts within both endothelial cells and beta cells to promote proliferation of developing beta cells.

Type 1 and type 2 diabetes result from an absolute or relative reduction in functional ß-cell mass. One approach to replacing lost ß-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance ß-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic ß cells that we previously showed is required for ß-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or ß cells results in decreased embryonic ß-cell proliferation, making CTGF unique as an identified ß cell-derived factor that regulates embryonic ß-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in ß-cell proliferation. Furthermore, CTGF overexpression in ß cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature ß cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature ß cells in directed differentiation or regeneration protocols.