Fetal Programming of the Endocrine Pancreas: Impact of a Maternal Low-Protein Diet on Gene Expression in the Perinatal Rat Pancreas.

In rats, the time of birth is characterized by a transient rise in beta cell replication, as well as beta cell neogenesis and the functional maturation of the endocrine pancreas. However, the knowledge of the gene expression during this period of beta cell expansion is incomplete. The aim was to characterize the perinatal rat pancreas transcriptome and to identify regulatory pathways differentially regulated at the whole organ level in the offspring of mothers fed a regular control diet (CO) and of mothers fed a low-protein diet (LP). We performed mRNA expression profiling via the microarray analysis of total rat pancreas samples at embryonic day (E) 20 and postnatal days (P) 0 and 2. In the CO group, pancreas metabolic pathways related to sterol and lipid metabolism were highly enriched, whereas the LP diet induced changes in transcripts involved in RNA transcription and gene regulation, as well as cell migration and apoptosis. Moreover, a number of individual transcripts were markedly upregulated at P0 in the CO pancreas: growth arrest specific 6 (Gas6), legumain (Lgmn), Ets variant gene 5 (Etv5), alpha-fetoprotein (Afp), dual-specificity phosphatase 6 (Dusp6), and angiopoietin-like 4 (Angptl4). The LP diet induced the downregulation of a large number of transcripts, including neurogenin 3 (Neurog3), Etv5, Gas6, Dusp6, signaling transducer and activator of transcription 3 (Stat3), growth hormone receptor (Ghr), prolactin receptor (Prlr), and Gas6 receptor (AXL receptor tyrosine kinase; Axl), whereas upregulated transcripts were related to inflammatory responses and cell motility. We identified differentially regulated genes and transcriptional networks in the perinatal pancreas. These data revealed marked adaptations of exocrine and endocrine in the pancreas to the low-protein diet, and the data can contribute to identifying novel regulators of beta cell mass expansion and functional maturation and may provide a valuable tool in the generation of fully functional beta cells from stem cells to be used in replacement therapy.

CTSH regulates ß-cell function and disease progression in newly diagnosed type 1 diabetes patients.

Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat ß-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower ß-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of ß-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic ß-cells, the target cells of the autoimmune assault.

Communication: Three-fold covariance imaging of laser-induced Coulomb explosions.

We apply a three-fold covariance imaging method to analyse previously acquired data [C. S. Slater et al., Phys. Rev. A 89, 011401(R) (2014)] on the femtosecond laser-induced Coulomb explosion of spatially pre-aligned 3,5-dibromo-3',5'-difluoro-4'-cyanobiphenyl molecules. The data were acquired using the "Pixel Imaging Mass Spectrometry" camera. We show how three-fold covariance imaging of ionic photofragment recoil trajectories can be used to provide new information about the parent ion's molecular structure prior to its Coulomb explosion. In particular, we show how the analysis may be used to obtain information about molecular conformation and provide an alternative route for enantiomer determination.

Dynamic stark control of torsional motion by a pair of laser pulses.

The torsional motion of a molecule composed of two substituted benzene rings, linked by a single bond, is coherently controlled by a pair of strong (3×10^{13} W cm^{-2}), nonresonant (800 nm) 200-fs-long laser pulses-both linearly polarized perpendicular to the single-bond axis. If the second pulse is sent at the time when the two benzene rings rotate toward (away from) each other the amplitude of the torsion is strongly enhanced (reduced). The torsional motion persists for more than 150 ps corresponding to approximately 120 torsional oscillations. Our calculations show that the key to control is the strong transient modification of the natural torsional potential by the laser-induced dynamic Stark effect.

Co-ordinated regulation of neurogenin-3 expression in the maternal and fetal pancreas during pregnancy.

OBJECTIVE: Several studies have shown increased beta cell mass during pregnancy in both rodents and humans. Proliferation of existing beta cells seems to be the predominant mechanism in rodents, whereas the mechanism in humans is unclear. We hypothesized that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved. SAMPLES: Pancreatic tissue from mice and rat and serum from pregnant women. METHOD: Morphometric analysis of pancreas of pregnant and nonpregnant mice was carried out by immunocytochemical staining for the neogenic marker neurogenin-3. Messenger RNA levels of neurogenin-3 and the transcription factor musculoaponeurotic fibrosarcoma oncogene family protein B in fetal rat pancreas cells, cultured with serum from pregnant women, were measured by quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: The number of neurogenin-3-positive cells present in pregnant mice was increased compared with nonpregnant mice. Neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA was detected in fetal rat pancreas exposed to serum from pregnant women. RESULTS: In pregnant mice we found a 3.6-fold increase in beta cell volume at day 18 compared with nonpregnant mice and a 3.5-fold increase in neurogenin-3 volume at day 14, mainly located in the acinar compartment where it was eightfold higher than in nonpregnant mice. In fetal rat pancreatic cells exposed to serum from pregnant women we found a marked increase in both neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA levels in fibroblast-like cells. CONCLUSION: These results suggest that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved in beta cell formation in both the maternal and fetal pancreas during pregnancy.

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy.

OBJECTIVE: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. SAMPLES: Serum samples from nonpregnant and pregnant women. METHODS: The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. MAIN OUTCOME MEASURES: Presence of circulating beta cell proliferating factors. RESULTS: Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. CONCLUSION: Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.

Impact of fetal and neonatal environment on beta cell function and development of diabetes.

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

Proinflammatory Cytokines Suppress Nonsense-Mediated RNA Decay to Impair Regulated Transcript Isoform Processing in Pancreatic ß-Cells.

Proinflammatory cytokines are implicated in pancreatic ß-cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of Nonsense-Mediated RNA Decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß-cells. A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3 or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. Gain- or loss-of function of two key NMD components UPF3B and UPF2 is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß-cells. These effects are found to involve ER stress and are associated with downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing, raises or lowers Cyt-induced cell death, respectively, in EndoC-ßH3 cells, and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increase alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in extracellular matrix (ECM) including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitises ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signalling, potentially serving as a protective response against Cyt-induced NMD component expression. Our findings highlight the central importance of RNA turnover in ß-cell responses to inflammatory stress.

Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic ß cells.

Introduction: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells. Methods: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Results: Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression. Conclusion: Our findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

Impulsive laser induced alignment of molecules dissolved in helium nanodroplets.

We show that a 450 fs nonresonant, moderately intense, linearly polarized laser pulse can induce field-free molecular axis alignment of methyliodide (CH(3)I) molecules dissolved in a helium nanodroplet. Time-resolved measurements reveal rotational dynamics much slower than that of isolated molecules and absence of the sharp transient alignment recurrences characteristic of gas phase molecules. Our results presage a range of new opportunities for exploring both molecular dynamics in a dissipative environment and the properties of He nanodroplets.

Mixed-field orientation of molecules without rotational symmetry.

The mixed-field orientation of an asymmetric-rotor molecule with its permanent dipole moment nonparallel to the principal axes of polarizability is investigated experimentally and theoretically. We find that for the typical case of a strong, nonresonant laser field and a weak static electric field complete 3D orientation is induced if the laser field is elliptically polarized and if its major and minor polarization axes are not parallel to the static field. For a linearly polarized laser field solely the dipole moment component along the most polarizable axis of the molecule is relevant resulting in 1D orientation even when the laser polarization and the static field are nonparallel. Simulations show that the dipole moment component perpendicular to the most-polarizable axis becomes relevant in a strong dc electric field combined with the laser field. This offers an alternative approach to 3D orientation by combining a linearly polarized laser field and a strong dc electric field arranged at an angle equal to the angle between the most polarizable axis of the molecule and its permanent dipole moment.

MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion.

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

Making the best of mixed-field orientation of polar molecules: a recipe for achieving adiabatic dynamics in an electrostatic field combined with laser pulses.

We have experimentally and theoretically investigated the mixed-field orientation of rotational-state-selected OCS molecules and achieved strong degrees of alignment and orientation. The applied moderately intense nanosecond laser pulses are long enough to adiabatically align molecules. However, in combination with a weak dc electric field, the same laser pulses result in nonadiabatic dynamics of the mixed-field orientation. These observations are fully explained by calculations employing both adiabatic and nonadiabatic (time-dependent) models.

Control and femtosecond time-resolved imaging of torsion in a chiral molecule.

We study how the combination of long and short laser pulses can be used to induce torsion in an axially chiral biphenyl derivative (3,5-difluoro-3',5'-dibromo-4'-cyanobiphenyl). A long, with respect to the molecular rotational periods, elliptically polarized laser pulse produces 3D alignment of the molecules, and a linearly polarized short pulse initiates torsion about the stereogenic axis. The torsional motion is monitored in real-time by measuring the dihedral angle using femtosecond time-resolved Coulomb explosion imaging. Within the first 4 picoseconds (ps), torsion occurs with a period of 1.25 ps and an amplitude of 3° in excellent agreement with theoretical calculations. At larger times, the quantum states of the molecules describing the torsional motion dephase and an almost isotropic distribution of the dihedral angle is measured. We demonstrate an original application of covariance analysis of two-dimensional ion images to reveal strong correlations between specific ejected ionic fragments from Coulomb explosion. This technique strengthens our interpretation of the experimental data.

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic ß-cells.

Fatty acid-induced damage in pancreatic ß-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve ß-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP2, FATP1 and FATP4 were unchanged. RNAi against FAT/CD36 decreased fatty acid-induced apoptosis. Over-expression of constitutively active STAT5 was able to mimic hGH's suppression of FAT/CD36 expression, whereas dominant negative STAT5 was unable to block the effect of hGH indicating that STAT5 did not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect ß-cells against fatty acid-induced damages.

Stark-selected beam of ground-state OCS molecules characterized by revivals of impulsive alignment.

We make use of an inhomogeneous electrostatic dipole field to impart a quantum-state-dependent deflection to a pulsed beam of OCS molecules, and show that those molecules residing in the absolute ground state, X(1)Σ(+), |00(0)0>, J = 0, can be separated out by selecting the most deflected part of the molecular beam. Past the deflector, we irradiate the molecular beam by a linearly polarized pulsed nonresonant laser beam that impulsively aligns the OCS molecules. Their alignment, monitored via velocity-map imaging, is measured as a function of time, and the time dependence of the alignment is used to determine the quantum state composition of the beam. We find significant enhancements of the alignment ( = 0.84) and of state purity (>92%) for a state-selected, deflected beam compared with an undeflected beam.

CXCL12/SDF-1 over-expression in human insulinomas and its biological relevance.

This study was performed on the basis of previously obtained investigative gene array data concerning the over-expression of CXCL12/SDF-1 in human insulinomas versus human pancreatic islet preparations. The presence of CXCL12/SDF-1 was studied by RT-qPCR in human insulinomas (n=8) versus pancreatic islets (n=3), and was found to be significantly up-regulated in the former (p<0.012). The mRNA data were confirmed by immunostaining and confocal microscopy of human normal pancreatic islets, which showed the absence of CXCL12 protein and high expression in insulinoma tissue. Individual human insulinoma cells at cytospins stained positive for CXCL12 in the paranuclear region. These morphological data were extended by consecutive immunoblotting for cell-compartment-specific marker proteins of fractions obtained by sucrose gradient fractionation using Rin-5F insulinoma cells. CXCL12-containing fractions were positive for the membrane marker NSF but negative for SNAP-25 and appeared at a lighter density in the gradient than heavy insulin granules, suggesting packaging in specific granules different from insulin. In order to determine the biological relevance of the protein in insulinomas, we investigated the colony-forming potential of human CXCL12 stable-transfected rat Rin-5F insulinoma cells. These clones secreted human CXCL12 and contained 50-1000-fold higher copy numbers compared to its endogenous rat homologue. In colony-forming assays, these transfectant clones developed greater colony numbers, which were larger than wild-type and sham transfectants. To elucidate the mechanism of action, we identified a CXCL12 transfectant-specific increase in the pro-survival factor Mn-SOD, which is considered important for the inactivation of reactive oxygen species, thereby prolonging cell survival. These data demonstrate the importance of CXCL12 in the tumor biology of insulinoma.

Laser-induced 3D alignment and orientation of quantum state-selected molecules.

A strong inhomogeneous static electric field is used to spatially disperse a rotationally cold supersonic beam of 2,6-difluoroiodobenzene molecules according to their rotational quantum state. The molecules in the lowest-lying rotational states are selected and used as targets for 3-dimensional alignment and orientation. The alignment is induced in the adiabatic regime with an elliptically polarized, intense laser pulse and the orientation is induced by the combined action of the laser pulse and a weak static electric field. We show that the degree of 3-dimensional alignment and orientation is strongly enhanced when rotational state-selected molecules, rather than molecules in the original molecular beam, are used as targets.

Quantum-state selection, alignment, and orientation of large molecules using static electric and laser fields.

Supersonic beams of polar molecules are deflected using inhomogeneous electric fields. The quantum-state selectivity of the deflection is used to spatially separate molecules according to their quantum state. A detailed analysis of the deflection and the obtained quantum-state selection is presented. The rotational temperatures of the molecular beams are determined from the spatial beam profiles and are all approximately 1 K. Unprecedented degrees of laser-induced alignment (=0.972) and orientation of iodobenzene molecules are demonstrated when the state-selected samples are used. Such state-selected and oriented molecules provide unique possibilities for many novel experiments in chemistry and physics.

Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated ß-cell maturation.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing ß-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat ß-cell line INS-1E. Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E ß-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E ß-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal ß-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3. These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate ß-cell maturation. This study indicates a functional role for TFF3 in pancreatic ß-cell maturation in the perinatal period, which is altered by low protein diet during gestation.