Adipose tissue macrophages (ATM) of obese patients are releasing increased levels of prolactin during an inflammatory challenge: a role for prolactin in diabesity?

BACKGROUND: Obesity, characterized by low grade inflammation, induces adipose tissue macrophage (ATM) infiltration in white adipose tissue (AT) in both humans and rodents, thus contributing to insulin resistance. Previous studies have shown altered prolactin secretion in obesity, however, studies linking ATM infiltration and prolactin (PRL) secretion to the pathogenesis of the metabolic syndrome, obesity and diabetes are lacking. METHODS/RESULTS: In vivo, qPCR and Western blot analysis demonstrated that prolactin expression was increased in AT of obese rats and also in human AT from obese, obese pre-diabetic and obese diabetic compared to lean counterparts. Immunohistochemistry of obese rat and human AT sections demonstrated a specific expression of prolactin in macrophages. In vitro, we demonstrated that hyperglycemia and inflammation stimulated macrophages (human THP-1 cell line and sorted rat ATM) to express PRL, when challenged with different glucose concentrations with or without IL1ß. In in vivo and in vitro experiments, we assessed the expression of Pit-1 (PRL-specific transcription factor) and found that its expression was parallel to PRL expression. CONCLUSIONS: In this study, we show that rodent and human macrophages synthesize prolactin in response to inflammation and high glucose concentrations. GENERAL SIGNIFICANCE: Our data shed new light on the potential role of macrophages in the physiopathology of diabesity via the PRL expression and on its expression mechanism and regulation.

The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic beta cells are killed by infiltrating immune cells and by cytokines released by these cells. Signaling events occurring in the pancreatic beta cells are decisive for their survival or death in diabetes. We have used RNA sequencing (RNA-seq) to identify transcripts, including splice variants, expressed in human islets of Langerhans under control conditions or following exposure to the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ). Based on this unique dataset, we examined whether putative candidate genes for T1D, previously identified by GWAS, are expressed in human islets. A total of 29,776 transcripts were identified as expressed in human islets. Expression of around 20% of these transcripts was modified by pro-inflammatory cytokines, including apoptosis- and inflammation-related genes. Chemokines were among the transcripts most modified by cytokines, a finding confirmed at the protein level by ELISA. Interestingly, 35% of the genes expressed in human islets undergo alternative splicing as annotated in RefSeq, and cytokines caused substantial changes in spliced transcripts. Nova1, previously considered a brain-specific regulator of mRNA splicing, is expressed in islets and its knockdown modified splicing. 25/41 of the candidate genes for T1D are expressed in islets, and cytokines modified expression of several of these transcripts. The present study doubles the number of known genes expressed in human islets and shows that cytokines modify alternative splicing in human islet cells. Importantly, it indicates that more than half of the known T1D candidate genes are expressed in human islets. This, and the production of a large number of chemokines and cytokines by cytokine-exposed islets, reinforces the concept of a dialog between pancreatic islets and the immune system in T1D. This dialog is modulated by candidate genes for the disease at both the immune system and beta cell level.

Diabetes during pregnancy influences Hofbauer cells, a subtype of placental macrophages, to acquire a pro-inflammatory phenotype.

Growing evidence indicates that maternal pathophysiological conditions, such as diabetes, influence fetal growth and could program metabolic disease in adulthood. Placental cells, particularly Hofbauer cells (HBCs), which are placental macrophages characterized by an anti-inflammatory profile (M2), can sense the modified maternal environment. The goal of this study was to investigate the direct effect of hyperglycemia on HBCs. We studied, at mRNA and protein levels, some markers of M2 and M1 (pro-inflammatory) macrophages in placentae from control and diabetic patients to assess the balance between pro- and anti-inflammatory macrophages: an imbalance of M2 to M1 macrophages has been observed in humans. We used pregnant rats, receiving a single injection of streptozotocin (STZ), as a model of maternal diabetes. We noticed a M2-to-M1 macrophage unbalance as we observed in human. An in vitro model of isolated rat HBCs was used to identify the direct effects of high glucose. We found that high glucose stimulation activated genes belonging to TLR (Toll-Like Receptor)-dependent inflammatory pathways. Moreover, the HBCs stimulated by high glucose switched their M2 profile towards M1, with increased expression of pro-inflammatory cytokines and markers. We also noticed that the oxidative-stress pathway was activated in response to high glucose driven by Hif-1α. In this study, we demonstrated that diabetes/hyperglycemia affect the anti-inflammatory profile of HBCs, by stimulating these cells to acquire an inflammatory profile leading to adverse consequences for the fetal-placental-maternal axis.

Thymus-specific deletion of insulin induces autoimmune diabetes.

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic beta-cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire-expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the beta-cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID-TEC mice for insulin-deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. beta-cell-specific autoimmune destruction was observed, as well as islet-specific T cell infiltration. The presence of insulin-specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti-insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes-resistant HLA class II alleles. ID-TEC mice could serve as a new model for studying this pathology.

Essential roles of insulin expression in Aire+ tolerogenic dendritic cells in maintaining peripheral self-tolerance of islet ß-cells.

Anti-insulin autoimmunity is one of the primary forces in initiating and progressing ß-cell destruction in type 1 diabetes. While insulin expression in thymic medullary epithelial cells has been shown to be essential for establishing ß-cell central tolerance, the function of insulin expression in antigen-presenting cells (APCs) of hematopoietic lineage remains elusive. With a Cre-lox reporter approach, we labeled Aire-expressing cells with enhanced yellow fluorescent proteins, and found that insulin expression in the spleen was restricted predominantly to a population of Aire(+)CD11c(int)B220(+) dendritic cells (DCs). Targeted insulin deletion in APCs failed to induce anti-islet autoimmunity in B6 mice. In contrast, elevated levels of T cell infiltration into islets were observed in B6(g7) congenic mice when insulin was specifically deleted in their CD11c-expressing DCs (B6(g7)·CD11c-ΔIns mice). Thus, insulin expression in BM-derived, Aire(+) tolerogenic DCs may play an essential role to prevent the activation and expansion of insulin-reactive T cells in the periphery.

Mung bean nuclease mapping of RNAs 3' end.

A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.

A homemade device for linear sucrose gradients.

We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials--a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained.

A new method for the mapping of 5' ends of RNAs.

In this article, we describe a new procedure to map 5' ends of RNAs. The procedure consists in the use of specific RNase H digestion of a hybrid formed by the RNA and a complementary DNA oligonucleotide. Northern blot hybridization of the resulting RNA fragment allows an accurate measurement of its length. Although we generally use this procedure as a control of previously performed primer extension analyses, the absence of nonspecific bands, which often occur in primer extensions on RNA templates with extended secondary structures, suggests that our method may be preferable when these difficult templates are analyzed.

Increasing voltage gradient electrophoresis of DNA.

We developed a method which allows electrophoretic fractionation of DNA in an agarose matrix according to an increasing current gradient, using a previously designed [R. Barbieri, V. Izzo, M.A. Costa, G. Giudice, G. Duro, Anal. Biochem. 212 (1993) 168; M.R. Asaro, V. Izzo, R. Barbieri, J. Chromatogr. A 855 (1999) 723] voltage gradient apparatus. This method allows the separation of different DNA fragments by increasing the distances of the components fractionated in the gel, revealing small differences in the length of different DNA components.

Long noncoding RNAs are dynamically regulated during ß-cell mass expansion in mouse pregnancy and control ß-cell proliferation in vitro.

Pregnancy is associated with increased ß-cell proliferation driven by prolactin. Long noncoding RNAs (lncRNA) are the most abundant RNA species in the mammalian genome, yet, their functional importance is mainly elusive. AIMS/HYPOTHESIS: This study tests the hypothesis that lncRNAs regulate ß-cell proliferation in response to prolactin in the context of ß-cell mass compensation in pregnancy. METHODS: The expression profile of lncRNAs in mouse islets at day 14.5 of pregnancy was explored by a bioinformatics approach, further confirmed by quantitative PCR at different days of pregnancy, and islet specificity was evaluated by comparing expression in islets versus other tissues. In order to establish the role of the candidate lncRNAs we studied cell proliferation in mouse islets and the MIN6 ß-cell line by EdU incorporation and cell count. RESULTS: We found that a group of lncRNAs is differentially regulated in mouse islets at 14.5 days of pregnancy. At different stages of pregnancy, these lncRNAs are dynamically expressed, and expression is prolactin dependent in mouse islets and MIN6 cells. One of those lncRNAs, Gm16308 (Lnc03), is dynamically regulated during pregnancy, prolactin-dependent and islet-enriched. Silencing Lnc03 in primary ß-cells and MIN6 cells inhibits, whereas over-expression stimulates, proliferation even in the absence of prolactin, demonstrating that Lnc03 regulates ß-cell growth. CONCLUSIONS/INTERPRETATION: During pregnancy mouse islet proliferation is correlated with dynamic changes of lncRNA expression. In particular, Lnc03 regulates mouse ß-cell proliferation and may be a crucial component of ß-cell proliferation in ß-cell mass adaptation in both health and disease.

Characterization of two alternative Interleukin(IL)-10 5'UTR mRNA sequences, induced by lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells.

IL-10 production shows a broad-spectrum of individual response, suggesting a genetic component of approximately 75%. Different polymorphisms located close to, or within the IL-10 gene has been demonstrated to influence its transcription rate whereas the post-transcriptional regulation of IL-10 production has not well elucidated. The main responsible elements at this control level are both the 5'- and 3'-untranslated regions (UTR's) of mRNAs, and as the 3'-UTR regions are mainly involved in the stability and decay rate of mRNAs, the 5'-UTR regions mediate the binding rate of the molecule with ribosomal 40S subunit as a cis-acting element. Herein are report data on the identification of two IL10 mRNA that differ by the length of respective 5'UTR regions (160 and 288 nucleotides, respectively; EMBL accession nrs: EU751618 and EU751619) produced after stimulation of human blood samples with bacterial lipopolysaccharide (LPS). The longer 5'UTR is constitutively expressed in unstimulated PBMC cells cultured at 37 degrees C for 24h, while in LPS stimulated cells an additional IL-10 mRNA molecule, containing a shorter 5'UTR, is synthesized. RNADRAW software (http://www.rnadraw.com/) analysis have indicated that the secondary structures of the shorter 5'UTR IL-10 mRNA region is more available for the binding to the 40S ribosomal subunit. In conclusion, our data seem to suggest that LPS could influence the post-transcriptional control of IL-10 production inducing an alternative mRNA immediately available in response to the inflammatory stimulation.

Orthogonal electrophoretic fractionation of DNA in agarose gels.

We developed an electrophoretic procedure, using Voltage Gradient Gel Electrophoresis (VGGE), which allows to obtain both an improvement of the resolution power of the system in orthogonal fractionation of DNA and, mainly, an about fourfold enhancement of hybridization signals in Southern blotting applications.

Chromosomal localization and molecular characterization of three different 5S ribosomal DNA clusters in the sea urchin Paracentrotus lividus.

In this paper the chromosomal localization and molecular cloning and characterization of three 5S rDNA clusters of 700 bp (base pairs), 900 bp, and 950 bp in the sea urchin Paracentrotus lividus are reported. Southern blot hybridization demonstrated the existence of three 5S rDNA repeats of differing length in the P. lividus genome. Fluorescence in situ hybridization analysis, performed in parallel on both haploid and diploid metaphases and interphase nuclei using different 5S rDNA units as probes, localized these 5S rDNA clusters in 3 different pairs of P. lividus chromosomes. This is the first complete gene mapping not only in a sea urchin but also in the phylum of echinoderms as a whole.

Improved resolution power of electrophoretic fractionation of DNA using a voltage gradient "up and down" application.

The improved resolution power of electrophoretic fractionation of DNA in a wide range of molecular masses is demonstrated using an "up and down" application of voltage gradient gel electrophoresis (VGGE). This application also allows separation of different DNA fragments which are poorly fractionated in conventional electrophoresis.