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Through its impact on photosynthesis and morphogenesis, light is the environmental factor that most affects plant architecture. Using light rather than chemicals to manage plant architecture could reduce the impact on the environment. However, the understanding of how light modulates plant architecture is still poor and further research is needed. To address this question, we examined the development of two rose cultivars, Rosa hybrida'Radrazz' and Rosa chinensis'Old Blush', cultivated under two light qualities. Plants were grown from one-node cuttings for 6 weeks under white or blue light at equal photosynthetic efficiencies. While plant development was totally inhibited in darkness, blue light could sustain full development from bud burst until flowering. Blue light reduced the net CO2 assimilation rate of fully expanded leaves in both cultivars, despite increasing stomatal conductance and intercellular CO2 concentrations. In 'Radrazz', the reduction in CO2 assimilation under blue light was related to a decrease in photosynthetic pigment content, while in both cultivars, the chl a/ b ratio increased. Surprisingly, blue light could induce the same organogenetic activity of the shoot apical meristem, growth of the metamers and flower development as white light. The normal development of rose plants under blue light reveals the strong adaptive properties of rose plants to their light environment. It also indicates that photomorphogenetic processes can all be triggered by blue wavelengths and that despite a lower assimilation rate, blue light can provide sufficient energy via photosynthesis to sustain normal growth and development in roses. [ABSTRACT FROM AUTHOR]

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention., Competing Interests: Declaration of competing interest No conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types., (© 2023. The Author(s).)

Loss of pancreatic β-cell mass is deleterious for type 2 diabetes patients since it reduces insulin production, critical for glucose homeostasis. The main research axis developed over the last few years was to generate new pancreatic β-cells or to transplant pancreatic islets as occurring for some specific type 1 diabetes patients. We evaluate here a new paradigm consisting in preservation of β-cells by prevention of human islet amyloid polypeptide (hIAPP) oligomers and fibrils formation leading to pancreatic β-cell death. We review the hIAPP physiology and the pathology that contributes to β-cell destruction, deciphering the various cellular steps that could be involved. Recent progress in understanding other amyloidosis such as Aβ, Tau, α-synuclein or prion, involved in neurodegenerative processes linked with inflammation, has opened new research lines of investigations to preserve neuronal cells. We evaluate and estimate their transposition to the pancreatic β-cells preservation. Among them is the control of reactive oxygen species (ROS) production occurring with inflammation and the possible implication of the mitochondrial translocator protein as a diagnostic and therapeutic target. The present review also focuses on other amyloid forming proteins from molecular to physiological and physiopathological points of view that could help to better decipher hIAPP-induced β-cell death mechanisms and to prevent hIAPP fibril formation., (Copyright © 2022. Published by Elsevier B.V.)

Glucocorticoids (GCs) are steroids secreted by the adrenal cortex under the hypothalamic-pituitary-adrenal axis control, one of the major neuro-endocrine systems of the organism. These hormones are involved in tissue repair, immune stability, and metabolic processes, such as the regulation of carbohydrate, lipid, and protein metabolism. Globally, GCs are presented as 'flight and fight' hormones and, in that purpose, they are catabolic hormones required to mobilize storage to provide energy for the organism. If acute GC secretion allows fast metabolic adaptations to respond to danger, stress, or metabolic imbalance, long-term GC exposure arising from treatment or Cushing's syndrome, progressively leads to insulin resistance and, in fine, cardiometabolic disorders. In this review, we briefly summarize the pharmacological actions of GC and metabolic dysregulations observed in patients exposed to an excess of GCs. Next, we describe in detail the molecular mechanisms underlying GC-induced insulin resistance in adipose tissue, liver, muscle, and to a lesser extent in gut, bone, and brain, mainly identified by numerous studies performed in animal models. Finally, we present the paradoxical effects of GCs on beta cell mass and insulin secretion by the pancreas with a specific focus on the direct and indirect (through insulin-sensitive organs) effects of GCs. Overall, a better knowledge of the specific action of GCs on several organs and their molecular targets may help foster the understanding of GCs' side effects and design new drugs that possess therapeutic benefits without metabolic adverse effects.

Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic β cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic β-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death., Competing Interests: Declaration of competing interest No conflict of interest to declare., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Both type 1 and type 2 diabetes are characterized by deficient insulin secretion and decreased β-cell mass. Thus, regenerative strategies to increase β-cell mass need to be developed. To characterize mechanisms of β-cell plasticity, we studied a model of severe insulin resistance in the adult mouse and defined how β-cells adapt. Chronic corticosterone (CORT) treatment was given to adult mice and led to rapid insulin resistance and adaptive increased insulin secretion. Adaptive and massive increase of β-cell mass was observed during treatment up to 8 weeks. β-Cell mass increase was partially reversible upon treatment cessation and reinduced upon subsequent treatment. β-Cell neogenesis was suggested by an increased number of islets, mainly close to ducts, and increased Sox9 and Ngn3 mRNA levels in islets, but lineage-tracing experiments revealed that neoformed β-cells did not derive from Sox9- or Ngn3-expressing cells. CORT treatment after β-cell depletion partially restored β-cells. Finally, β-cell neogenesis was shown to be indirectly stimulated by CORT because serum from CORT-treated mice increased β-cell differentiation in in vitro cultures of pancreatic buds. Altogether, the results present a novel model of β-cell neogenesis in the adult mouse and identify the presence of neogenic factors in the serum of CORT-treated mice., (© 2018 by the American Diabetes Association.)

Background: Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation., Results: In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates., Conclusions: Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.

Type 2 diabetes mellitus is characterized histopathologically by the presence of fibrillary amyloid deposits in the pancreatic islets of Langerhans. Human islet amyloid polypeptide (hIAPP), the 37-residue pancreatic hormone, is the major constituent of these amyloid deposits. The propensity of IAPP to form amyloid fibrils is strongly dependent on its primary sequence. An intriguing example is His at residue 18. Although H18 is located outside the amyloidogenic region, it has been suggested that this residue and its charge state play an important role in the kinetics of conformational changes and fibril formation as well as in mediating cell toxicity. To gain more insight into the importance of this residue, we have synthesized four analogues (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and we performed a full biophysical study on the properties of these peptides. Kinetic experiments as monitored by thioflavin-T fluorescence, transmission electron microscopy, circular dichroism and cell toxicity assays revealed that all variants are less fibrillogenic and less toxic than native hIAPP both at neutral pH and at low pH. This demonstrates that the effect of H18 in native IAPP is not simply determined by its charge state, but rather that residue 18 is important for specific intra- and intermolecular interactions that occur during fibril formation and that may involve charge, size and hydrophobicity. Furthermore, our results indicate that H18R-IAPP has a strong inhibiting effect on native hIAPP fibril formation. Together these results highlight the large impact of modifying a single residue outside the amyloidogenic domain on fibril formation and cell toxicity induced by IAPP, opening up new avenues for design of inhibitors or modulators of IAPP aggregation., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

Intestine contributes to energy homeostasis through the absorption, metabolism, and transfer of nutrients to the organism. We demonstrated previously that hepatocyte nuclear receptor-4α (HNF-4α) controls intestinal epithelium homeostasis and intestinal absorption of dietary lipids. HNF-4γ, the other HNF-4 form highly expressed in intestine, is much less studied. In HNF-4γ knockout mice, we detect an exaggerated insulin peak and improvement in glucose tolerance during oral but not intraperitoneal glucose tolerance tests, highlighting the involvement of intestine. Moreover, the enteroendocrine L-type cell lineage is modified, as assessed by the increased expression of transcription factors Isl1, Foxa1/2, and Hnf4a, leading to an increase of both GLP-1-positive cell number and basal and stimulated GLP-1 plasma levels potentiating the glucose-stimulated insulin secretion. Using the GLP-1 antagonist exendin (9-39), we demonstrate a direct effect of GLP-1 on improved glucose tolerance. GLP-1 exerts a trophic effect on pancreatic β-cells, and we report an increase of the β-cell fraction correlated with an augmented number of proliferative islet cells and with resistance to streptozotocin-induced diabetes. In conclusion, the loss of HNF-4γ improves glucose homeostasis through a modulation of the enteroendocrine cell lineage., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and β cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced β cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic β cell development and insulin secretion.

This is essential to understand habitat selection by wildlife to manage habitats and populations. Studying the annual use of aquatic habitats provides information on how to manage wetlands for waterfowl, and to predict possible detrimental effects associated with extended usage by these birds. This is particularly important for species like the mute swan (Cygnus olor Gmelin), given its recent dramatic demographic expansion, causing concern in both Europe and America. We studied the extent of usage (swan.days.ha(-1)) of habitat patches by mute swans in a heterogeneous and fluctuating fishpond landscape. We assessed seasonal differences of swan usage of fishponds, annual variation for a given fishpond, and determined which habitat factors drive swan usage over the year. The seasonal use pattern was regular: a similar proportion of fishponds was used heavily, moderately or lightly in all seasons. Flocking throughout the year and breeding during summer were associated with heavy use of fishponds, i.e. large number of swan.days.ha(-1). Flocking on some fishponds during several successive seasons demonstrated that some waterbody provide valuable habitats over time for swans. However, swans did not use individual fishponds to the same extent each season, mostly depending on the fluctuating ecological requirements of swans and variation in habitat properties. Agricultural practices on fishponds drastically affected swan usage during autumn and winter: formerly dried fishponds were used preferentially once reflooded. The specific agricultural crops used during the drought period had no influence though. The large-sized fishponds and fishponds within a dense network of waterbody were the most heavily used by swans throughout the year. Our results may thus be helpful to predict and prevent possible habitat damage by swans. They also provide information on habitats that are valuable for waterfowl species in general, by using mute swans as a proxy for waterfowl requirements., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Pancreatic exocrine and endocrine cells develop during embryonic life from endodermal progenitors. This process depends on activation of a hierarchy of transcription factors. Although information is available regarding the mesodermal signals controlling pancreas development, little is known about the role of environmental factors such as nutrients, including glucose, that also may impact development. Previously, we showed that glucose plays an important and specific role in beta cell development by activating the transition of Neurogenin3-positive endocrine progenitors into beta cells. Here, we examined the implication of glucose metabolism and more precisely the role of the hexosamine biosynthesis pathway (HBP) to understand the mechanisms by which glucose regulates beta cell development. We have established an in vitro model of endocrine and exocrine cells development from embryonic day 13.5 rat pancreases in a manner that replicates in vivo pancreas development perfectly. Using this model, we tested the effect of selective inhibitors and activators of the HBP and found that the HBP has a modest effect on cell proliferation and exocrine cell differentiation. On the other hand, beta cell development is tightly controlled by the HBP. Specifically, HBP activators increase beta cell development, whereas inhibitors repress such development. Importantly, both the HBP and glucose control the same steps in beta cell development.

Understanding pancreatic development is important for at least three reasons: first, from a cognitive point of view, to understand the development of a complex organ, the pancreas; next, because it is now clear that abnormal pancreatic development can give rise to specific forms of diabetes in humans; and finally, because, if we want to define new treatments for diabetes based on cell therapy or regenerative medicine, we will have to understand in detail how beta-cells develop. In the present paper, we summarize what we currently know concerning pancreatic development and concentrate on some intercellular and environmental signals controlling pancreatic development.

Mature pancreatic cells develop during embryonic life from endodermal progenitors, and this developmental process depends on activation of a hierarchy of transcription factors. While information is available on mesodermal signals controlling pancreas development, little is known about environmental factors, such as the levels of nutrients including glucose, that may control this process. Here, we studied the effects of glucose on pancreatic cells development. We used an in vitro model where both endocrine and acinar cells develop from early pancreatic and duodenal homeobox-1 (PDX1)-positive embryonic pancreatic progenitors. We first showed that glucose does not have a major effect on global pancreatic cell proliferation, survival, and acinar cell development. On the other hand, glucose controlled both alpha and beta cell development. Specifically, the surface occupied by insulin-positive cells was 20-fold higher in pancreases cultured in presence than in absence of glucose, and this effect was dose-dependent over the range 0.5-10 mm. Glucose did not appear to control beta cell development by activating the proliferation of early progenitors or beta cells themselves but instead tightly regulated cell differentiation. Thus, glucose did not modify the pattern of expression of Neurogenin3, the earliest marker of endocrine progenitor cells, but was necessary for the expression of the transcription factor NeuroD, a direct target of Neurogenin3 known to be important for proper pancreatic endocrine cell development. We conclude that glucose interferes with the pancreatic endocrine cells development by regulating the transition between Ngn3 and upstream NeuroD.

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.

The transcription factor PDX-1 (pancreatic and duodenal homeobox-1) is essential for pancreatic development and the maintainence of expression of islet beta-cell-specific genes. In an previous study [Rafiq, Kennedy and Rutter (1998) J. Biol. Chem. 273, 23241-23247] we demonstrated that PDX-1 may be activated at elevated glucose concentrations by translocation from undefined binding sites in the cytosol and nuclear membrane into the nucleoplasm. In the present study, we show that PDX-1 interacts directly and specifically in vitro with the nuclear import receptor family member, importin beta1, and that this interaction is mediated by the PDX-1 homeodomain (amino acids 146-206). Demonstrating the functional importance of the PDX-1-importin beta1 interaction, microinjection of MIN6 beta-cells with anti-(importin beta1) antibodies blocked both the nuclear translocation of PDX-1, and the activation by glucose (30 mM versus 3 mM) of the pre-proinsulin promoter. However, treatment with extracts from pancreatic islets incubated at either low or high glucose concentrations had no impact on the ability of PDX-1 to interact with importin beta1 in vitro. Furthermore, importin beta1 also interacted with SREBP1c (sterol-regulatory-element-binding protein 1c) in vitro, and microinjection of importin beta1 antibodies blocked the activation by glucose of SREBP1c target genes. Since the subcellular distribution of SREBP1c is unaffected by glucose, these findings suggest that a redistribution of importin beta1 is unlikely to explain the glucose-stimulated nuclear uptake of PDX-1. Instead, we conclude that the uptake of PDX-1 into the nucleoplasm, as glucose concentrations increase, may be mediated by release of the factor both from sites of retention in the cytosol and from non-productive complexes with importin beta1 at the nuclear membrane.

We studied the role of the karyopherin alpha2 nuclear import carrier (also known as importin alpha2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin alpha2 accumulated massively in the cytoplasm within minutes of glucose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin alpha1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin alpha2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin alpha2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyopherin alpha2 and the stimulation of glucose-induced gene transcription were simultaneously lost between passages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin alpha2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

A novel protein was cloned from a rat liver cDNA library by interaction with the liver glucokinase. This protein contained 339 residues and possessed a canonical consensus sequence for a dual specificity phosphatase. The recombinant protein was able to dephosphorylate phosphotyrosyl and phosphoseryl/threonyl substrates. We called this protein the glucokinase-associated phosphatase (GKAP). The GKAP partially dephosphorylated the recombinant glucokinase previously phosphorylated, in vitro, by protein kinase A. The GKAP fused with green fluorescent protein was located in the cytosol, where glucokinase phosphorylates glucose, and not in the nucleus where the glucokinase is retained inactive by the glucokinase regulatory protein. More importantly, the GKAP accelerated the glucokinase activity in a dose-dependent manner and with a stoichiometry compatible with a physiological mechanism. This strongly suggested that the interaction between GKAP and glucokinase had a functional significance. The cloning of this novel protein with a dual specificity phosphatase activity allows the description of a possible new regulatory step in controlling the glycolysis flux.

Genetically hypertensive rats receiving a high sodium content diet develop, within weeks of their birth, major alterations of tissues and blood vessels. The purpose of this study was to evaluate the effects of cicletanine, a synthetic antihypertensive drug, on the progress of genetic hypertension. Iffa Credo SHR-SP male rats aged 11 weeks were divided into 4 groups. One group was used as control and 3 groups were treated with oral cicletanine in doses of 10, 30 and 90 mg/kg/day respectively. The control group showed a high mortality rate due to a significant decrease of weight gain and a highly significant increase of blood pressure, these changes being associated with lesions of tissues and vessels in the brain, heart and kidneys. A curative treatment with cicletanine improved these parameters and was accompanied by good tissue preservation. The curative effect of cicletanine seems to be due to an increase in endogenous prostaglandin synthesis.